Protein-based streptococcus pneumoniae vaccines

ABSTRACT

Vaccine compositions and methods for protecting a mammalian subject against infection with  S. pneumoniae  are disclosed. These vaccine compositions include as the active ingredient a purified preparation of the cell wall protein ABC transporter substrate-binding protein having the Accession No. NP_344690 and the amino acid sequence set forth in SEQ ID NO: 32, optionally together with one or more pharmaceutically acceptable adjuvants.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a division of U.S. application Ser. No. 13/734,350filed Jan. 4, 2013, which is a continuation-in-part of U.S. applicationSer. No. 12/435,781 filed May 5, 2009, abandoned, which is acontinuation-in-part of U.S. application Ser. No. 12/363,383 filed Jan.30, 2009, abandoned, which is a division of U.S. application Ser. No.10/953,513 filed Sep. 30, 2004, U.S. Pat. No. 7,504,110, which is acontinuation-in-part of International application no. PCT/IL2003/000271filed Apr. 1, 2003, which claims the benefit of U.S. provisionalapplication No. 60/368,981 filed Apr. 2, 2002.

FIELD OF THE INVENTION

The present invention relates to vaccine compositions and methods forprotecting against infection with Streptococcus pneumoniae. Morespecifically, the present invention provides vaccine compositionscomprising S. pneumoniae cell wall or cell membrane proteins associatedwith an age-dependent immune response.

BACKGROUND OF THE INVENTION

The Gram-positive bacterium Streptococcus pneumoniae is a major cause ofdisease, suffering and death worldwide. Diseases caused by infectionwith this agent include otitis media, pneumonia, bacteremia, sepsis andmeningitis. In some cases, infected individuals may become asymptomaticcarriers of S. pneumoniae, thereby readily allowing the rapid spread ofthis infective agent throughout the population. In view of the seriousconsequences of infection with S. pneumoniae, as well as its rapidspread within and between populations, there is an urgent need for safe,effective vaccination regimes. Current methods of vaccination are basedon inoculation of the subject with polysaccharides obtained from thecapsules of S. pneumoniae. While these polysaccharide-based vaccinepreparations have been found to be reasonably efficacious when used toprevent infection in adult populations, they are significantly lessuseful in the treatment of young children (under two years of age) andthe elderly. One commonly-used capsular polysaccharide 23-valentvaccine, for example, has been found to be only 60% effective inpreventing S. pneumoniae invasive disease in elderly subjects andcompletely incapable of yielding neither long-term memory (Hammitt etal., 2011, Vaccine 29:2287-2295) nor clinically-useful antibodyresponses in the under-two age group (Shapiro E. D. et al., 1991, N.Engl. J. Med. 325: 1453-1460).

In an attempt to increase the immunogenicity of these vaccines, variouscompositions comprising capsular polysaccharides that have beenconjugated with various carrier proteins and combined with adjuvant havebeen used. The resulting so-called conjugate vaccines (CV) currentlyinclude 10-13 serotypes. Although vaccines of this type constitute animprovement in relation to the un-conjugated polysaccharide vaccines,they have not overcome the problem of coverage, since they are effectiveagainst only about 10% of the 92 known capsular serotypes. Consequently,upon vaccination, pneumococcal carriage and repopulation with serotypesnot present in the vaccine occurs (Dagan, 2009, Vaccine 27 Suppl3:C22-24).

In the cases of certain other bacteria of pathogenic importance forhuman and other mammalian species, vaccines comprising immunogenicvirulence proteins are currently being developed. Such protein-basedvaccines should be of particular value in the case of vulnerablesubjects such as very young children, in view of the fact that suchsubjects are able to produce antibodies against foreign proteins.Unfortunately, very little is known of the molecular details of the lifecycle of S. pneumoniae, or of the nature of role of the variousvirulence factors which are known or thought to be involved in targetingand infection of susceptible hosts.

Several publications describe and characterize specific S. pneumoniaeproteins. For example, U.S. Pat. No. 5,958,734, U.S. Pat. No. 5,976,840,U.S. Pat. No. 6,165,760 and U.S. Pat. No. 6,300,119 disclose S.pneumoniae GtS polypeptides of various lengths, polynucleotides encodingthem and methods for producing such polypeptides by recombinanttechniques. WO 02/077021 the sequences of about 2,500 S. pneumoniaegenes and their corresponding amino acid sequences from type 4 strainthat were identified in silico. U.S. Pat. No. 6,699,703 and itscounterparts discloses about 2600 S. pneumoniae polypeptides and methodsfor producing such polypeptides by recombinant techniques, compositionscomprising same and methods of use in the preparation of a vaccine. WO98/23631 relates to 111 Streptococcal polynucleotides identified ashaving a GUG start codon, which encodes a Val residue, to polypeptidesencoded by such polynucleotides, and to their production and uses. WO02/083833 discloses 376 S. pneumoniae polypeptide antigens which aresurface localized, membrane associated, secreted or exposed on thebacteria, for preparation of a diagnostic kit and or vaccine. Althoughsuggested in part of the publications, no working examples for the useof the proteins as antigens in the production of a vaccine wereprovided. Furthermore, none of these references disclose or suggest thatuse of selected protein antigens which do no elicit immune response ininfants and in elderly, improve the outcome of vaccination against S.pneumoniae.

Phosphoenolpyruvate protein phosphotransferase (PPP, also known as PtsA)is an intracellular protein that belongs to the sugar phosphotransferasesystem (PTS) and is also localized to the bacterial cell wall. In thecytoplasm PPP belongs to the group of phosphtransferase systems (PTS)responsible for carbohydrate internalization, which occurs concurrentlywith their phosphorylation. The phosphorylation of the membrane-spanningenzyme is dependent upon a group of proteins that sequentially transfera phosphate group to this enzyme. PPP is a cytoplasmic protein thatcatalyzes the initial step in this process by transferring a phosphategroup from phosphoenolpyruvate to a histidine in another enzyme, HrP inthis system (Saier, M. H., Jr. & Reizer, J., 1992, J Bacteriol174:1433-1438).

There is an unmet need to provide protein-based vaccine compositionswhich overcome the problems and drawbacks of currently availablevaccines, by being effective against a wide range of different S.pneumoniae serotypes, and capable of protecting all age groups includinginfants and elderly.

SUMMARY OF THE INVENTION

It has now been found that it is possible to protect individuals againstinfection with S. pneumoniae by means of administering to saidindividuals a vaccine composition comprising one or more proteinsisolated from the outer layers of the aforementioned bacteria and/or oneor more immunonologically-active fragments, derivatives or modificationsthereof. Unexpectedly, it was found that a defined set of proteins,associated with age-dependent immunity, are effective in vaccinecompositions against a wide range of different S. pneumoniae serotypes,and in all age groups, including those age groups that do not produceanti-S. pneumoniae antibodies following vaccination withpolysaccharide-based compositions, or those resulting in a shift inserotype distribution towards those pneumococcal capsularpolysaccharides that are not present in the vaccine. These age groupsinclude infants aged 0-4 years and elderly. Thus, the use of the set ofantigens in accordance with the principle of the invention overcomes thedisadvantages of known vaccines.

It is now disclosed that the antibody response to S. pneumoniae proteinsincreases with age in infants and this increase correlates negativelywith morbidity. Antibodies to S. pneumoniae protein antigens develop inhumans during the asymptomatic carriage and invasive disease. Infantsbelow two years of age who are at most risk from pneumococcal infectionsdo not respond efficiently to currently available polysaccharide-basedvaccination. It is now unexpectedly shown, using sera longitudinallycollected from healthy children, exposed to bacterial infections thatthere is an age-dependent enhancement of the antibody response tocertain S. pneumoniae surface protein antigens, while in most otherproteins there is no enhancement of immunogenicity during the checkedtime period. This enhancement, with age, of antibody responses against aset of specific pneumococcal surface proteins is implicated in thedevelopment of natural immunity and was used in the present invention toidentify candidate antigens (herein “age dependent proteins”) for use inimproved vaccine compositions effective in all age groups, includinginfants, immunocompromized subjects and elderly.

In elderly subjects capsular polysaccharide based vaccines are only 60%effective in preventing S. pneumoniae invasive disease. An elderlysubject should be vaccinated at least once in five years and thevaccination efficacy is reduced in each repeated vaccination. Theprotein-based vaccines of the present invention, which are T-celldependent antigens, are expected to be more effective than thepolysaccharide-based vaccines in elderly subjects.

The present invention provides a method for protecting individualsagainst infection with S. pneumoniae by the use of a protein-basedvaccine.

The present invention further provides a protein-based vaccine that isprepared from at least one of a specific set of immunogenic cell walland/or cell membrane proteins of S. pneumoniae, having age-dependentimmune responses, or from one or more immunologically-active fragments,derivatives or modifications thereof.

According to one aspect of the present invention, a vaccine compositioncomprises as an active ingredient one or more isolated proteins selectedfrom one or more S. pneumoniae cell wall or cell membrane proteins orimmunologically-active protein fragments, derivatives or modificationsthereof, which are associated with an age-dependent immune response.According to preferred embodiments, this aspect of the invention saidthe age-dependent S. pneumoniae cell wall and/or cell membrane proteinis selected from the group consisting of: phosphoenolpyruvate proteinphosphotransferase (Accession No. NP_(—)345645, SEQ ID NO: 4);phosphoglucomutase/phosphomannomutase family protein (Accession No.NP_(—)346006, SEQ ID NO:5); trigger factor (Accession No. NP_(—)344923,SEQ ID NO: 6); elongation factor G/tetracycline resistance protein(tetO), (Accession No. NP_(—)344811, SEQ ID NO: 7); NADH oxidase(Accession No. NP_(—)345923, SEQ ID NO: 8); Aspartyl/glutamyl-tRNAamidotransferase subunit C (Accession No. NP_(—)344960, SEQ ID NO: 9);cell division protein FtsZ (Accession No. NP_(—)346105, SEQ ID NO: 10);L-lactate dehydrogenase (Accession No. NP_(—)345686, SEQ ID NO: 11);glyceraldehyde 3-phosphate dehydrogenase (GAPDH), (Accession No.NP_(—)346439, SEQ ID NO: 12); fructose-bisphosphate aldolase (AccessionNo. NP_(—)345117, SEQ ID NO: 13); UDP-glucose 4-epimerase (Accession No.NP_(—)346261, SEQ ID NO: 14); elongation factor Tu family protein(Accession No. NP_(—)358192, SEQ ID NO: 15); Bifunctional GMPsynthase/glutamine amidotransferase protein (Accession No. NP_(—)345899,SEQ ID NO: 16); glutamyl-tRNA synthetase (Accession No. NP_(—)346492,SEQ ID NO: 17); glutamate dehydrogenase (Accession No. NP_(—)345769, SEQID NO: 18); Elongation factor TS (Accession No. NP_(—)346622, SEQ ID NO:19); phosphoglycerate kinase (TIGR4) (Accession No. AAK74657, SEQ ID NO:20); 30S ribosomal protein 51 (Accession No. NP_(—)345350, SEQ ID NO:21); 6-phosphogluconate dehydrogenase (Accession No. NP_(—)357929, SEQID NO: 22); aminopeptidase C (Accession No. NP_(—)344819, SEQ ID NO:23); carbamoyl-phosphate synthase (large subunit) (Accession No.NP_(—)345739, SEQ ID NO: 24); PTS system, mannose-specific IIABcomponents (Accession No. NP_(—)344822, SEQ ID NO: 25); 30S ribosomalprotein S2 (Accession No. NP_(—)346623, SEQ ID NO: 26); dihydroorotatedehydrogenase 1B (Accession No. NP_(—)358460, SEQ ID NO: 27); aspartatecarbamoyltransferase catalytic subunit (Accession No. NP_(—)345741, SEQID NO: 28); elongation factor Tu (Accession No. NP_(—)345941, SEQ ID NO:29); Pneumococcal surface immunogenic protein A (PsipA) (Accession No.NP_(—)344634, SEQ ID NO: 30); phosphoglycerate kinase (R6) (AccessionNo. NP_(—)358035, SEQ ID NO: 31); ABC transporter substrate-bindingprotein (Accession No. NP_(—)344690, SEQ ID NO: 32); endopeptidase 0(Accession No. NP_(—)346087, SEQ ID NO: 33); Pneumococcal surfaceimmunogenic protein B (PsipB) (Accession No. NP_(—)358083, SEQ ID NO:34); Pneumococcal surface immunogenic protein C (PsipC) (Accession No.NP_(—)345081, SEQ ID NO: 35).

According to a particular embodiment, the vaccine composition comprisesthe age-dependent protein phosphoenolpyruvate protein phosphotransferase(PPP or rPtsA) of Accession No. NP_(—)345645, set forth in SEQ ID NO: 4,or a fragment or modification thereof wherein such fragment ormodification is capable of eliciting an immune response against S.pneumoniae.

According to other embodiments, the vaccine composition comprises atleast one age-dependent protein selected from the group consisting of:phosphoenolpyruvate protein phosphotransferase (PPP, Accession No.NP_(—)345644, SEQ ID NO: 4); Fructose-bisphosphate aldolase (NP 345117,SEQ ID NO: 13); Aminopeptidase C (NP_(—)344819, SEQ ID NO: 23); NADHoxidase (NOX, NP_(—)345923, SEQ ID NO: 8) and ABC transportersubstrate-binding protein (Accession No. NP_(—)344690, SEQ ID NO: 32).

According to some embodiments the one or more bacterial proteins of thevaccine are effective in all age groups, including those age groups thatdo not produce anti-S. pneumoniae antibodies following vaccination withpolysaccharide-based vaccines; or exposure to the bacteria.

According to one embodiment the age group comprises infants less thanfour years of age. According to another embodiment the age groupcomprises infants less than two years of age.

According to one embodiment the age group comprises elderly subjects.According to yet another embodiment the age group comprises childrenolder the 4 years of age and adult subjects.

According to another embodiment the age group comprisesimmunocompromised subjects.

The vaccine compositions of the present invention may also containother, non-immunologically-specific additives, diluents and excipients.For example, in many cases, the vaccine compositions of the presentinvention may contain, in addition to the S. pneumoniae cell-wall and/orcell-membrane protein(s), one or more adjuvants.

Pharmaceutically acceptable adjuvants include, but are not limited towater in oil emulsion, lipid emulsion, and liposomes. According tospecific embodiments the adjuvant is selected from the group consistingof: Montanide®, alum, muramyl dipeptide, Gelvac®, chitin microparticles,chitosan, cholera toxin subunit B, labile toxin, AS21A, ASO2V,Intralipid®, Lipofundin, Monophosphoryl lipid A; RIBI: monophosphoryllipid A with Mycobacterial cell wall components (muramy tri peptide),ISCOMs Immune stimulating complexes, CpG, and DNA vaccines such as pVAC.Also included are immune enhancers such as cytokines.

In some embodiments the vaccine composition is formulated forintramuscular, intranasal, oral, intraperitoneal, subcutaneous, topical,intradermal and transdermal delivery. In some embodiments the vaccine isformulated for intramuscular administration. In other embodiments thevaccine is formulated for oral administration. In yet other embodimentsthe vaccine is formulated for intranasal administration.

In one particularly preferred embodiment, the method of the presentinvention for protection of mammalian subjects against infection with S.pneumoniae comprises administering to a subject in need of suchprotection an effective amount of at least one cell wall and/or cellmembrane proteins associated with age-related immune response, and/orimmunogenically-active fragments, derivatives or modifications thereof,wherein said at least one protein is selected from the group consistingof: fructose-bisphosphate aldolase (FBA, NP_(—)345117, SEQ ID NO:13),Phosphoenolpyruvate protein phosphotransferase (PPP) NP_(—)345645 (SEQID NO:4), Glutamyl tRNA synthetase (GtS, NP_(—)346492, SEQ ID NO:17),NADH oxidase (NOX, NP_(—)345923, SEQ ID NO:8), Pneumococcal surfaceimmunogenic protein B (PsipB; NP_(—)358083, SEQ ID NO:34), triggerfactor (TF, NP_(—)344923, SEQ ID NO:6), FtsZ cell division protein(NP_(—)346105, SEQ ID NO:10), PTS system, mannose-specific IIABcomponents (PTS, NP_(—)344822, SEQ ID NO:25), and Elongation factor G(EFG, NP344811, SEQ ID NO:7).

According to a particular embodiment, the method comprisesadministration of the protein phosphoenolpyruvate proteinphosphotransferase (PPP or rPtsA) of Accession No. NP_(—)345645, setforth in SEQ ID NO: 4, or a fragment or modification thereof whereinsuch fragment or modification is capable of eliciting an immune responseagainst S. pneumoniae.

According to some embodiments at least one protein of the vaccinecomposition is an enzyme involved in glycolysis. According to a specificembodiment the at least one protein involved in glycolysis is selectedfrom the group consisting of: L-lactate dehydrogenase (SEQ ID NO: 11),UDP-glucose 4-epimerase (SEQ ID NO: 14), fructose-bisphosphate aldolase(SEQ ID NO: 13), glyceraldehyde-3-phosphate dehydrogenase (SEQ ID NO:12), phosphoglycerate kinase (SEQ ID NO: 31) and 6-phosphoglutamatedehydrogenase (SEQ ID NO: 22).

According to another embodiment at least one protein of the vaccinecomposition is an enzyme involved in protein synthesis. According to aspecific embodiment the protein involved in protein synthesis isglutamyl-tRNA amidotransferase (SEQ ID NO: 16) or glutamyl-tRNAsynthetase (SEQ ID NO: 17).

According to other embodiments at least one protein of the vaccinecomposition is an enzyme belonging to the other physiological pathwaysselected from: NADP glutamate dehydrogenase (NP_(—)345769),aminopeptidase C (Accession No. NP_(—)344819, SEQ ID NO: 23),carbamoylphosphate synthase (Accession No. NP_(—)345739, SEQ ID NO: 24),aspartate carbamoyltransferase (Accession No. NP_(—)345741, SEQ ID NO:28), NADH oxidase (NOX, Accession No. NP_(—)345923, SEQ ID NO: 8),Pneumococcal surface immunogenic protein B (PsipB, Accession No.NP_(—)358083, SEQ ID NO: 34); and pyruvate oxidase.

In some embodiments the cell wall and/or cell membrane proteins arelectins. According to specific embodiments the lectin proteins areselected from the group consisting of: Fructose-bisphosphate aldolase(NP 345117, SEQ ID NO:13); Aminopeptidase C (NP_(—)344819, SEQ IDNO:23).

According to some embodiments the S. pneumoniae proteins and/orfragments, derivatives or modifications thereof are lectins and thevaccine compositions comprising them are particularly efficacious in theprevention of localized S. pneumoniae infections. In one preferredembodiment, the localized infections are infections of mucosal tissue,particularly of nasal and other respiratory mucosa.

In alternative embodiments of the method of the invention, the cell walland/or cell membrane proteins are non-lectins.

In specific embodiments the non-lectin proteins are selected from thegroup consisting of: Phosphomannomutase (NP 346006, SEQ ID NO:5);Trigger factor (NP 344923, SEQ ID NO:6); NADH oxidase (NP 345923, SEQ IDNO:8); L-lactate dehydrogenase (NP 345686, SEQ ID NO:11); Glutamyl-tRNAsynthetase (NP 346492, SEQ ID NO:17).

According to other embodiments the S. pneumoniae proteins and/or theirfragments, derivatives or modifications used in the aforementionedmethods, compositions and vaccines are non-lectins, and the vaccinecompositions are particularly efficacious in the prevention of systemicS. pneumoniae infections.

In another preferred embodiment of the method of the invention, vaccinecomposition comprises at least one lectin protein and at least onenon-lectin protein.

The present invention is directed according to another aspect to amethod for preventing infection of mammalian subjects with S.pneumoniae, wherein said method comprises administering to a subject inneed of such treatment an effective amount of one or more S. pneumoniaecell wall and/or cell membrane proteins associated with age-relatedimmune response, and/or immunogenically-active fragments, derivatives ormodifications thereof, wherein said proteins are selected from the groupconsisting of: phosphoenolpyruvate protein phosphotransferase (AccessionNo. NP_(—)345645, SEQ ID NO:4); phosphoglucomutase/phosphomannomutasefamily protein (Accession No. NP_(—)346006, SEQ ID NO:5); trigger factor(Accession No. NP_(—)344923, SEQ ID NO:6); elongation factorG/tetracycline resistance protein (tetO), (Accession No. NP_(—)344811,SEQ ID NO:7); NADH oxidase (Accession No. NP_(—)345923, SEQ ID NO:8);Aspartyl/glutamyl-tRNA amidotransferase subunit C (Accession No.NP_(—)344960, SEQ ID NO:9); cell division protein FtsZ (Accession No.NP_(—)346105, SEQ ID NO:10); L-lactate dehydrogenase (Accession No.NP_(—)345686, SEQ ID NO:11); glyceraldehyde 3-phosphate dehydrogenase(GAPDH), (Accession No. NP_(—)346439, SEQ ID NO:12);fructose-bisphosphate aldolase (Accession No. NP_(—)345117, SEQ IDNO:13); UDP-glucose 4-epimerase (Accession No. NP_(—)346261, SEQ IDNO:14); elongation factor Tu family protein (Accession No. NP_(—)358192,SEQ ID NO:15); Bifunctional GMP synthase/glutamine amidotransferaseprotein (Accession No. NP_(—)345899, SEQ ID NO:16); glutamyl-tRNAsynthetase (Accession No. NP_(—)346492, SEQ ID NO:17); glutamatedehydrogenase (Accession No. NP_(—)345769, SEQ ID NO:18); Elongationfactor TS (Accession No. NP_(—)346622, SEQ ID NO:19); phosphoglyceratekinase (TIGR4) (Accession No. AAK74657, SEQ ID NO:20); 30S ribosomalprotein 51 (Accession No. NP_(—)345350, SEQ ID NO:21);6-phosphogluconate dehydrogenase (Accession No. NP_(—)357929, SEQ IDNO:22); aminopeptidase C (Accession No. NP_(—)344819, SEQ ID NO:23);carbamoyl-phosphate synthase (large subunit) (Accession No.NP_(—)345739, SEQ ID NO:24); PTS system, mannose-specific IIABcomponents (Accession No. NP_(—)344822, SEQ ID NO:25); 30S ribosomalprotein S2 (Accession No. NP_(—)346623, SEQ ID NO:26); dihydroorotatedehydrogenase 1B (Accession No. NP_(—)358460, SEQ ID NO:27); aspartatecarbamoyltransferase catalytic subunit (Accession No. NP_(—)345741, SEQID NO:28); elongation factor Tu (Accession No. NP_(—)345941, SEQ IDNO:29); Pneumococcal surface immunogenic protein A (PsipA) (AccessionNo. NP_(—)344634, SEQ ID NO:30); phosphoglycerate kinase (R6) (AccessionNo. NP_(—)358035, SEQ ID NO:31); ABC transporter substrate-bindingprotein (Accession No. NP_(—)344690, SEQ ID NO:32); endopeptidase 0(Accession No. NP_(—)346087, SEQ ID NO:33); Pneumococcal surfaceimmunogenic protein B (PsipB) (Accession No. NP_(—)358083, SEQ IDNO:34); Pneumococcal surface immunogenic protein C (PsipC) (AccessionNo. NP 345081, SEQ ID NO:35).

Vaccine compositions of the present invention can be administered to asubject in need thereof, prior to, during or after occurrence ofinfection or inoculation with S. pneumoniae.

The vaccine compositions of the present invention are administered,according to one embodiment by means of injection. According to someembodiments the injection route is selected from the group consistingof: intramuscular, intradermal or subcutaneous. According to otherembodiments the injection route is selected from intravenous andintraperitoneal. According to yet other embodiments the vaccinecompositions of the present invention are administered by nasal or oralroutes.

According to some embodiments the S. pneumoniae proteins and/orfragments, derivatives or modifications thereof are lectins and thevaccine compositions comprising them are particularly efficacious in theprevention of localized S. pneumoniae infections. In one preferredembodiment, the localized infections are infections of mucosal tissue,particularly of nasal and other respiratory mucosa.

According to other embodiments the S. pneumoniae proteins and/or theirfragments, derivatives or modifications used in the aforementionedmethods, compositions and vaccines are non-lectins, and the vaccinecompositions are particularly efficacious in the prevention of systemicS. pneumoniae infections.

In another preferred embodiment of the method of the invention, vaccinecomposition comprises at least one lectin protein and at least onenon-lectin protein.

In one preferred embodiment of the method of the invention, themammalian subject is a human subject.

The aforementioned vaccine compositions may clearly be used forpreventing infection of the mammalian subjects by S. pneumoniae.However, said vaccine composition is not restricted to this use alone.Rather it may be usefully employed to prevent infection by anyinfectious agent whose viability or proliferation may be inhibited bythe antibodies generated by a host in response to the inoculationtherein of the one or more S. pneumoniae proteins provided in saidcomposition.

According to some embodiments the vaccine compositions of the presentinvention inhibit S. pneumoniae adhesion to cells, for example to humanlung cells.

DNA vaccines comprising at least one polynucleotide sequence encodingage-dependent bacterial proteins according to the invention are alsowithin the scope of the present invention, as well as methods forprotecting a mammalian subject against infection with S. pneumoniaecomprising administering such polynucleotide sequence to a subject.According to one embodiment the present invention provides a vaccinecomposition comprising at least one polynucleotide sequence encoding aprotein selected from one or more S. pneumoniae cell wall or cellmembrane proteins or immunogenically-active protein fragments,derivatives or modifications thereof, which is associated with anage-dependent immune response. According to some embodiments the DNAvaccine composition further comprises at least one polynucleotidesequence encoding an adjuvant peptide or protein. According to apreferred embodiment a DNA vaccine according to the invention isadministered by intramuscular injection.

The present invention discloses, according to yet a further aspect, amethod for identifying bacterial proteins having age-dependentimmunogenicity. Identified age-dependent proteins can be used in vaccinecompositions against pathogens expressing said proteins.

According to certain embodiments, a method for identifying a bacterialprotein having age-dependent immunogenicity is provided the methodcomprises the steps of: providing an extract of the cell wall and/orcell membrane of the pathogen; separating the extract by2D-electrophoresis or micro-chromatography; blotting the protein extractto a matrix; probing the blots with sera collected longitudinally fromchildren at different ages; identifying the protein spots havingintensity increasing with age; thereby identifying a protein havingage-dependent immunogenicity.

According to some embodiments the protein extract is blotted onto apaper. According to other embodiments the proteins are identified usingMatrix Assisted Laser Desorption/Ionization mass spectrometry (MALDI-MS)technique.

According to some embodiments the pathogen is a bacterium. According tospecific embodiments the bacterium is S. pneumoniae and the sera arecollected from children aged 18, 30 and 42 months. According to anotherembodiment the pathogen is Streptococcus pyogenes.

All of the above and other characteristics and advantages of the presentinvention will be further understood from the following illustrative andnon-limitative examples of preferred embodiments thereof.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a photograph of a Western blot in which the sera of miceimmunized with (A) recombinant GAPDH and (B) recombinantfructose-bisphosphate aldolase are seen to recognize the correspondingnative proteins (CW) (in an electrophoretically-separated total cellwall protein preparation), and the corresponding recombinant protein(R).

FIG. 2 is a photograph of a Western blot in which the sera of miceimmunized with pVAC constructs containing the cDNA of S. pneumoniaefructose-bipshosphate aldolase (A) and GAPDH (B) are seen to recognizethe corresponding native proteins from electrophoretically-separatedtotal cell wall protein preparation. Sera obtained followingimmunization with the pVAC parental plasmid did not recognize either ofthe two proteins (C).

FIGS. 3A and 3B each shows a graph describing the ability of recombinantGAPDH (3B) and fructose-bisphosphate aldolase (3A) to elicit aprotective immune response to intraperitoneal and intranasal challengewith a lethal dose of S. pneumoniae in the mouse model system.

FIG. 4 is a photograph of a gel depicting the 297 base pair ALDO1-containing fragment of S. pneumoniae fructose bisphosphate aldolase.

FIG. 5 depicts an agarose gel separation of ALDO 1 and the pHAT vectorafter restriction by Kpn1 and SacI enzymes.

FIG. 6 is a photograph of an agarose gel showing the 297 by PCRamplification product (comprising ALDO 1) obtained from coloniestransformed with the pHAT/ALDO 1 construct.

FIG. 7A-E describes the vaccine potential of PPP. BALB/c mice wereimmunized SC with rPPP formulated with CFA (day 0) and IFA (days 14 &28), and followed for colonization (7A-7D) or mortality (7E) followingIN inoculation. (7A) strain WU2, 3 h (p<0.01), (7B) strain WU2 48 h(p<0.05), (7C) Strain D39, NP, 48 h (p<0.001), (7D) Strain D39, lung, 48h (p<0.007), (7E) Strain WU2, 7 days of observation for mortality(p<0.05).

FIG. 8 depicts the increased survival of mice following a lethalintranasal inoculation of mice following immunization with recombinantGlutamyl tRNA synthetase (rGtS).

FIG. 9 describes survival of mice following active immunization withrecombinant NADH oxidase (rNOX).

FIG. 10 survival of mice after passive IP immunization with: anti-rPsipBantiserum, control preimmune serum, or anti-non-lectin protein mixture(NL) serum. The mice were inoculated intraperitonealy with the antiserum24 and 3 hours prior to bacterial challenge.

FIG. 11 active immunization of mice with Trigger factor (TF) usingCFA/IFA/IFA immunization protocol in comparison to control (adjuvant)immunized animals.

FIG. 12 survival of mice following IP challenge with S. pneumoniae after1 hour neutralization with anti-FtsZ cell division protein (FtsZ)antiserum, preimmune serum or anti NL serum.

FIG. 13 survival of mice following IP challenge with S. pneumoniaeneutralized with anti-PTS system, mannose-specific IIAB components (PTS)antiserum, preimmune serum or NL serum.

FIG. 14 mice survival after active immunization with Elongation factor G(EFG) with Alum adjuvant in comparison to mice injected with adjuvantonly as control.

FIG. 15 reconfirms the age dependent recognition of GtS by sera obtainedlongitudinally from children attending day care centers and a serumobtained from an adult subject.

FIG. 16 reconfirms the age dependent recognition of NOX, using rNOX, bysera obtained longitudinally from children attending day care centers.

FIG. 17A-B demonstrates surface expression and conservation of PPP indifferent strains. 17A. Membrane and cell-wall (CW) protein fractionsfrom four clinical isolates were immunoblotted with mouse anti-PtsAantibodies; rPtsA positive control (upper lane). The membrane wasimmunoblotted with pre-immune serum as negative control (lower lane).17B. CW and cytoplasmic protein fractions immunoblotted with rabbitanti-FabD antibodies.

FIG. 18 reconfirms the age dependent immunogenicity of PPP. rPPP wasimmunoblotted with sera obtained from infants attending day care centersat (18A) 7, (18B) 12, (18C) 24, and (D) 38 months of age.

DETAILED DESCRIPTION OF PREFERRED EMBODIMENTS

As disclosed herein for the first time, specific pneumococcal surfaceproteins that exhibit age-dependent immunogenicity, which coincide withthe development of natural protective immunity. Proteins identifiedusing antibodies against these proteins, present in infant sera, elicita protective response against S. pneumoniae and can be used forprotection against infection with the bacteria. It is now shown thatproteins identified as exhibiting age-dependent immune response ininfants, or antibodies to such proteins were able to protect miceagainst infection with S. pneumoniae.

Vaccine compositions according to the present invention may be used forpreventing infection of the mammalian subjects by S. pneumoniae.However, said vaccine compositions may be also usefully employed toprevent adhesion of the bacteria to cells and to inhibit and reducebacterial load and bacterial carriage. It was shown (Daniely et al.,2006, Clin. Exp. Immunol. 144, 254-263; Mizrachi Nebenzahl et al., 2007,J. Infectious Diseases 196:945-53), that antibodies to proteinsidentified in the present application as possessing age-dependentimmunogenicity are capable of inhibiting S. pneumoniae adhesion to humanlung cells.

The immunologically variant capsular polysaccharides of S. pneumoniaeare used widely for the typing of clinical isolates. There are more than90 capsular serotypes and their prevalence among human isolates varieswith age, disease type and to some extent geographical origin. A23-valent capsular polysaccharide-based vaccine is licensed for use inadults, but it does not elicit an efficient antibody response orprotection in children under 2 years of age and immunocompromisedpatients. To overcome this lack of responsiveness to the T cellindependent polysaccharide antigens in young children the conjugatepneumococcal vaccines were developed. These vaccines consist of 7 to 13of the most prevalent S. pneumoniae capsular polysaccharides covalentlylinked to a protein carrier to stimulate T cell responses to thevaccine. These vaccines are highly effective in preventing invasivepneumococcal disease in infants but there are some drawbacks associatedwith the complexity of the manufacturing process that increase costs andthe limited number of various capsular polysaccharides that can beincluded in the vaccine. Vaccination with conjugate pneumococcalvaccines has recently been shown to result in a shift in serotypedistribution toward those pneumococcal capsular polysaccharides that arenot present in the vaccine. In addition, geographical variations in theprevalence of clinically important serotypes of S. pneumoniae weredescribed. These concerns combined with the increasing antibioticresistance are driving research efforts to develop a wide rangepneumococcal vaccine that is immunogenic in all age groups and broadlycross-protective against all or most serotypes. In addition proteins areT cell dependent antigen and are more likely to induce long lastingimmunological memory.

The reasons to longitudinally start collecting sera from day-carechildren who are frequently exposed to S. pneumoniae, aiming to identifyprotein antigens involved in the development of natural immunity to S.pneumoniae, at 18 months of age were:

i. During gestation maternal IgG antibodies cross the placenta and inthe initial months of life these maternal antibodies are protecting theinfants.ii. Starting at 6 months of age the levels of the maternal antibodiesdecline and a gradual increase in the infants' antibodies start toappear.iii. Children are most susceptible to S. pneumoniae infections between5-35 months of age. The first decrease in their susceptibility can beobserved at between 12-23 months of age however the most significantdecrease occurs between 24-35 months of age. It is assumed that naturalstrong immune response to a protein (for example Pyruvate oxidase andEnolase table 2), preceding this time period is not sufficient toprotect children from S. pneumoniae infections. Therefore these proteinswhich did not elicit natural protection against the bacteria although animmune response against them is high in young infants are notage-dependent.

Immunodeficiency comprises a highly variable group of diseases. Whileprimary immunodeficiency result from genetic alteration in genesaffecting the immune response, acquired immunodeficiency result frominfection with pathogens that affects the immune system (such as HIV-1).Other conditions that may cause diminution of the immune response andincrease susceptibility to infections include malnutrition and diseasessuch as cancer. Most of the immunocompromised patients have acquiredimmunodeficiency. Malfunction of the immune system may stem from eitherlack of or the existence of dysfunctional B cells or T cells ormacrophages. In other cases immunodeficiency may result in loss ofimmune memory cells. Antibody deficiencies comprise the most commontypes of primary immune deficiencies in human subjects. Such patientsare highly susceptible to encapsulated bacterial infections. Forexample, patients that have B cell immunodeficiency could benefit fromvaccination with the proteins of the present invention, which are T celldependent antigens. Patients that demonstrated loss of immune memory,including HIV-1 patients, could also benefit from vaccination with thecompositions of the present invention.

Thus it was suspected that the most significant development of naturalimmunity occurs after two years of age and it was chosen to encompassthis period in the attempt to identify proteins that the immuneresponses to them increase with age during this period.

Vaccination of infants in the first year of age with the age-dependentbacterial proteins of the invention is expected to elicit protectiveimmune responses to the bacteria, simulating the development of naturalprotective immunity that occurs at an older age.

Vaccination protects individuals (and by extension, populations) fromthe harmful effects of pathogenic agents, such as bacteria, by inducinga specific immunological response to said pathogenic agents in thevaccinated subject.

Vaccines are generally, but not exclusively, administered by means ofinjection, generally by way of the intramuscular, intradermal orsubcutaneous routes. Some vaccines may also be administered by theintravenous, intraperitoneal, nasal or oral routes.

The S. pneumoniae-protein containing preparations of the invention canbe administered as either single or multiple doses of an effectiveamount of said protein. The term “effective amount” is used herein toindicate that the vaccine is administered in an amount sufficient toinduce or boost a specific immune response, such that measurable amounts(or an increase in the measurable amounts) of one or more antibodiesdirected against the S. pneumoniae proteins used may be detected in theserum or plasma of the vaccinated subject. The precise weight of proteinor proteins that constitutes an “effective amount” will depend upon manyfactors including the age, weight and physical condition of the subjectto be vaccinated. The precise quantity also depends upon the capacity ofthe subject's immune system to produce antibodies, and the degree ofprotection desired. Effective dosages can be readily established by oneof ordinary skill in the art through routine trials establishing doseresponse curves. However, for the purposes of the present invention,effective amounts of the compositions of the invention can vary from0.01-1,000 μg/ml per dose, more preferably 0.1-500 μg/ml per dose,wherein the usual dose size is 1 ml.

The vaccine compositions of the present invention, capable of protectingsubject from infection or inoculation with S. pneumoniae can beadministered to a subject in need thereof, prior to, during or afteroccurrence of infection or inoculation with the bacteria.

In general, the vaccines of the present invention would normally beadministered parenterally, by the intramuscular, intravenous,intradermal or subcutaneous routes, either by injection or by a rapidinfusion method. Compositions for parenteral administration includesterile aqueous or non-aqueous solutions, suspensions, and emulsions.Examples of non-aqueous solvents are propylene glycol, polyethyleneglycol, vegetable oils such as olive oil, and injectable organic esterssuch as ethyl oleate. Besides the abovementioned inert diluents andsolvents, the vaccine compositions of the invention can also includeadjuvants, wetting agents, emulsifying and suspending agents, orsweetening, flavoring, or perfuming agents.

The vaccines of the present invention will generally comprise aneffective amount of one or more S. pneumoniae proteins as the activecomponent, suspended in an appropriate vehicle. In the case ofintranasal formulations, for example, said formulations may includevehicles that neither cause irritation to the nasal mucosa norsignificantly disturb ciliary function. Diluents such as water, aqueoussaline may also be added. The nasal formulations may also containpreservatives including, but not limited to, chlorobutanol andbenzalkonium chloride. A surfactant may be present to enhance absorptionof the subject proteins by the nasal mucosa. An additional mode ofantigen delivery may include an encapsulation technique, which involvescomplex coacervation of gelatin and chondroitin sulfate (Azhari R, LeongK W. 1991. Complex coacervation of chondroitin sulfate and gelatin andits use for encapsulation and slow release of a model protein. Proc.Symp. Control. Rel. 18: 617; Brown K E, Leong K, Huang C H, Dalal R,Green G D, Haimes H B, Jimenez P A, Bathon J. 1998. Gelatin chondroitin6-sulfate microspheres for delivery of therapeutic proteins to thejoint. Arthritis Rheum 41: 2185-2195).

DEFINITIONS

The term “immunologically-active” is used herein in ordinary sense torefer to an entity (such as a protein or its fragment or derivative)that is capable of eliciting an immune response when introduced into ahost subject.

The term “immunogenic protein” according to the present inventiondenotes a bacterial protein that was identified by antibodies present inhuman sera. “Antigenicity” refers to the ability of the bacterialprotein to produce antibodies against it in the host. The term“age-related immune response” or “age dependent protein” (as usedthroughout this application) indicates that the ability of subjects toproduce antibodies to the bacterial protein or proteins, causing saidimmune response, increases with age. In the case of human subjects, saidability is measured over a time scale beginning with neonates and endingat approximately four years of age and adults. In non-human mammaliansubjects, the “age-related immune response” is measured over an agerange extending from neonates to an age at which the immune system ofthe young mammal is at a stage of development comparable to that of apre-puberty human child and adults.

It is to be noted that in the context of the present invention, theterms “fragments”, “derivatives” and “modifications” are to beunderstood as follows:

“Fragment”: a less than full length portion, or linked portions, of thenative sequence of the protein in question, wherein the sequence of saidportion is essentially unchanged as compared to the relevant part of thesequence of the native protein.

“Derivative”: a full length, and a less than full length portion of thenative sequence of the protein in question, wherein either the sequencefurther comprises (at its termini and/or within said sequence itself)non-native amino acid sequences, i.e. sequences which do not form partof the native protein in question. The term “derivative” also includeswithin its scope molecular species produced by conjugating chemicalgroups to the amino residue side chains of the native proteins orfragments thereof, wherein said chemical groups do not form part of thenaturally-occurring amino acid residues present in said native proteins.

“Modification”: a full length protein or less than full length portionthereof comprising at least one amino acid residue which is not nativelypresent in the same location in the sequence of said protein, which havebeen introduced as a consequence of mutation of the native sequence (byeither random or site-directed processes), by chemical modification orby chemical synthesis.

The term “infection” as used herein in the present application refers toa state in which disease-causing S. pneumoniae have invaded, colonized,spread, adhered, disseminated or multiplied in body cells or tissues.This term encompass the term “inoculation”, namely the state in whichthe bacteria colonized the nasopharynx but there are no infectionsymptoms yet.

The term “lectins” is used hereinabove and hereinbelow to indicateproteins having the ability to bind specifically to polysaccharides oroligosaccharides. Conversely, the term “non-lectins” is used to refer toproteins lacking the aforementioned saccharide-binding property, or toproteins which do not bind the saccharides tested in the presentapplication.

Vaccine Formulation

The vaccines of the present invention comprise at least one bacterialprotein exhibiting an age-dependent increase antibody response ininfants, fragment, derivative or modification of said bacterial protein,and optionally, an adjuvant. Formulation can contain a variety ofadditives, such as adjuvant, excipient, stabilizers, buffers, orpreservatives. The vaccine can be formulated for administration in oneof many different modes.

In preferred embodiment, the vaccine is formulated for parenteraladministration, for example intramuscular administration. According toyet another embodiment the administration is orally.

According to yet another embodiment the administration is intradermal.Needles specifically designed to deposit the vaccine intradermally areknown in the art as disclosed for example in 6,843,781 and 7,250,036among others. According to other embodiments the administration isperformed with a needleless injector.

According to one embodiment of the invention, the vaccine isadministered intranasally. The vaccine formulation may be applied to thelymphatic tissue of the nose in any convenient manner. However, it ispreferred to apply it as a liquid stream or liquid droplets to the wallsof the nasal passage. The intranasal composition can be formulated, forexample, in liquid form as nose drops, spray, or suitable forinhalation, as powder, as cream, or as emulsion.

In another embodiment of the invention, administration is oral and thevaccine may be presented, for example, in the form of a tablet orencased in a gelatin capsule or a microcapsule.

The formulation of these modalities is general knowledge to those withskill in the art.

Liposomes provide another delivery system for antigen delivery andpresentation. Liposomes are bilayered vesicles composed of phospholipidsand other sterols surrounding a typically aqueous center where antigensor other products can be encapsulated. The liposome structure is highlyversatile with many types range in nanometer to micrometer sizes, fromabout 25 nm to about 500 μm. Liposomes have been found to be effectivein delivering therapeutic agents to dermal and mucosal surfaces.Liposomes can be further modified for targeted delivery by for example,incorporating specific antibodies into the surface membrane, or alteredto encapsulate bacteria, viruses or parasites. The average survival timeor half life of the intact liposome structure can be extended with theinclusion of certain polymers, for example polyethylene glycol, allowingfor prolonged release in vivo. Liposomes may be unilamellar ormultilamellar.

The vaccine composition may be formulated by: encapsulating an antigenor an antigen/adjuvant complex in liposomes to formliposome-encapsulated antigen and mixing the liposome-encapsulatedantigen with a carrier comprising a continuous phase of a hydrophobicsubstance. If an antigen/adjuvant complex is not used in the first step,a suitable adjuvant may be added to the liposome-encapsulated antigen,to the mixture of liposome-encapsulated antigen and carrier, or to thecarrier before the carrier is mixed with the liposome-encapsulatedantigen. The order of the process may depend on the type of adjuvantused. Typically, when an adjuvant like alum is used, the adjuvant andthe antigen are mixed first to form an antigen/adjuvant complex followedby encapsulation of the antigen/adjuvant complex with liposomes. Theresulting liposome-encapsulated antigen is then mixed with the carrier.The term “liposome-encapsulated antigen” may refer to encapsulation ofthe antigen alone or to the encapsulation of the antigen/adjuvantcomplex depending on the context. This promotes intimate contact betweenthe adjuvant and the antigen and may, at least in part, account for theimmune response when alum is used as the adjuvant. When another is used,the antigen may be first encapsulated in liposomes and the resultingliposome-encapsulated antigen is then mixed into the adjuvant in ahydrophobic substance.

In formulating a vaccine composition that is substantially free ofwater, antigen or antigen/adjuvant complex is encapsulated withliposomes and mixed with a hydrophobic substance. In formulating avaccine in an emulsion of water-in-a hydrophobic substance, the antigenor antigen/adjuvant complex is encapsulated with liposomes in an aqueousmedium followed by the mixing of the aqueous medium with a hydrophobicsubstance. In the case of the emulsion, to maintain the hydrophobicsubstance in the continuous phase, the aqueous medium containing theliposomes may be added in aliquots with mixing to the hydrophobicsubstance.

In all methods of formulation, the liposome-encapsulated antigen may befreeze-dried before being mixed with the hydrophobic substance or withthe aqueous medium as the case may be. In some instances, anantigen/adjuvant complex may be encapsulated by liposomes followed byfreeze-drying. In other instances, the antigen may be encapsulated byliposomes followed by the addition of adjuvant then freeze-drying toform a freeze-dried liposome-encapsulated antigen with externaladjuvant. In yet another instance, the antigen may be encapsulated byliposomes followed by freeze-drying before the addition of adjuvant.Freeze-drying may promote better interaction between the adjuvant andthe antigen resulting in a more efficacious vaccine.

Formulation of the liposome-encapsulated antigen into a hydrophobicsubstance may also involve the use of an emulsifier to promote more evendistribution of the liposomes in the hydrophobic substance. Typicalemulsifiers are well-known in the art and include mannide oleate(Arlacel™ A), lecithin, Tween™ 80, Spans™ 20, 80, 83 and 85. Theemulsifier is used in an amount effective to promote even distributionof the liposomes. Typically, the volume ratio (v/v) of hydrophobicsubstance to emulsifier is in the range of about 5:1 to about 15:1.

Microparticles and nanoparticles employ small biodegradable sphereswhich act as depots for vaccine delivery. The major advantage thatpolymer microspheres possess over other depot-effecting adjuvants isthat they are extremely safe and have been approved by the Food and DrugAdministration in the US for use in human medicine as suitable suturesand for use as a biodegradable drug delivery system (Langer R. Science.1990; 249(4976):1527-33). The rates of copolymer hydrolysis are verywell characterized, which in turn allows for the manufacture ofmicroparticles with sustained antigen release over prolonged periods oftime (O'Hagen, et al., Vaccine, 1993; 11:965-9).

Parenteral administration of microparticles elicits long-lastingimmunity, especially if they incorporate prolonged releasecharacteristics. The rate of release can be modulated by the mixture ofpolymers and their relative molecular weights, which will hydrolyze overvarying periods of time. Without wishing to be bound to theory, theformulation of different sized particles (1 μm to 200 μm) may alsocontribute to long-lasting immunological responses since large particlesmust be broken down into smaller particles before being available formacrophage uptake. In this manner a single-injection vaccine could bedeveloped by integrating various particle sizes, thereby prolongingantigen presentation and greatly benefiting livestock producers.

In some applications an adjuvant or excipient may be included in thevaccine formulation. Montanide™ (Incomplete Freund's adjuvant) and alumfor example, are preferred adjuvants for human use. The choice of theadjuvant will be determined in part by the mode of administration of thevaccine. A preferred mode of administration is intramuscularadministration. Another preferred mode of administration is intranasaladministration. Non-limiting examples of intranasal adjuvants includechitosan powder, PLA and PLG microspheres, QS-21, ASO2A, calciumphosphate nanoparticles (CAP); mCTA/LTB (mutant cholera toxin E112K withpentameric B subunit of heat labile enterotoxin), and detoxified E. Coliderived labile toxin.

The adjuvant used may also be, theoretically, any of the adjuvants knownfor peptide- or protein-based vaccines. For example: inorganic adjuvantsin gel form (aluminium hydroxide/aluminium phosphate, Warren et al.,1986; calcium phosphate, Relyvelt, 1986); bacterial adjuvants such asmonophosphoryl lipid A (Ribi, 1984; Baker et al., 1988) and muramylpeptides (Ellouz et al., 1974; Allison and Byars, 1991; Waters et al.,1986); particulate adjuvants such as the so-called ISCOMS(“immunostimulatory complexes”, Mowat and Donachie, 1991; Takahashi etal., 1990; Thapar et al., 1991), liposomes (Mbawuike et al. 1990;Abraham, 1992; Phillips and Emili, 1992; Gregoriadis, 1990) andbiodegradable microspheres (Marx et al., 1993); adjuvants based on oilemulsions and emulsifiers such as IFA (“Incomplete Freund's adjuvant”(Stuart-Harris, 1969; Warren et al., 1986), SAF (Allison and Byars,1991), saponines (such as QS-21; Newman et al., 1992), squalene/squalane(Allison and Byars, 1991); synthetic adjuvants such as non-ionic blockcopolymers (Hunter et al., 1991), muramyl peptide analogs (Azuma, 1992),synthetic lipid A (Warren et al., 1986; Azuma, 1992), syntheticpolynucleotides (Harrington et al., 1978) and polycationic adjuvants (WO97/30721).

Adjuvants for use with immunogens of the present invention includealuminum or calcium salts (for example hydroxide or phosphate salts). Aparticularly preferred adjuvant for use herein is an aluminum hydroxidegel such as Alhydrogel™. Calcium phosphate nanoparticles (CAP) is anadjuvant being developed by Biosante, Inc (Lincolnshire, Ill.). Theimmunogen of interest can be either coated to the outside of particles,or encapsulated inside on the inside (He et al., 2000, Clin. Diagn. Lab.Immunol., 7, 899-903).

Another adjuvant for use with an immunogen of the present invention isan emulsion. A contemplated emulsion can be an oil-in-water emulsion ora water-in-oil emulsion. In addition to the immunogenic chimer proteinparticles, such emulsions comprise an oil phase of squalene, squalane,peanut oil or the like as are well known, and a dispersing agent.Non-ionic dispersing agents are preferred and such materials includemono- and di-C₁₂-C₂₄-fatty acid esters of sorbitan and mannide such assorbitan mono-stearate, sorbitan mono-oleate and mannide mono-oleate.

Such emulsions are for example water-in-oil emulsions that comprisesqualene, glycerol and a surfactant such as mannide mono-oleate(Arlacel™ A), optionally with squalane, emulsified with the chimerprotein particles in an aqueous phase. Alternative components of theoil-phase include alpha-tocopherol, mixed-chain di- and tri-glycerides,and sorbitan esters. Well-known examples of such emulsions includeMontanide™ ISA-720, and Montanide™ ISA 703 (Seppic, Castres, France.Other oil-in-water emulsion adjuvants include those disclosed in WO95/17210 and EP 0 399 843.

The use of small molecule adjuvants is also contemplated herein. Onetype of small molecule adjuvant useful herein is a 7-substituted-8-oxo-or 8-sulfo-guanosine derivative described in U.S. Pat. No. 4,539,205,U.S. Pat. No. 4,643,992, U.S. Pat. No. 5,011,828 and U.S. Pat. No.5,093,318. 7-allyl-8-oxoguanosine(loxoribine) has been shown to beparticularly effective in inducing an antigen-(immunogen-) specificresponse.

A useful adjuvant includes monophosphoryl lipid A (MPL®), 3-deacylmonophosphoryl lipid A (3D-MPL®), a well-known adjuvant manufactured byCorixa Corp. of Seattle, formerly Ribi Immunochem, Hamilton, Mont. Theadjuvant contains three components extracted from bacteria:monophosphoryl lipid (MPL) A, trehalose dimycolate (TDM) and cell wallskeleton (CWS) (MPL+TDM+CWS) in a 2% squalene/Tween™ 80 emulsion. Thisadjuvant can be prepared by the methods taught in GB 2122204B.

Other compounds are structurally related to MPL® adjuvant calledaminoalkyl glucosamide phosphates (AGPs) such as those available fromCorixa Corp under the designation RC-529™ adjuvant{2-[(R)-3-tetra-decanoyloxytetradecanoylamino]-ethyl-2-deoxy-4-O-phosphon-o-3-O—[(R)-3-tetradecanoyloxytetra-decanoyl]-2-[(R)-3-tetra-decanoyloxytet-radecanoyl-amino]-p-D-glucopyranosidetriethylammonium salt}. An RC-529 adjuvant is available in a squaleneemulsion sold as RC-529SE and in an aqueous formulation as RC-529AFavailable from Corixa Corp. (see, U.S. Pat. No. 6,355,257 and U.S. Pat.No. 6,303,347; U.S. Pat. No. 6,113,918; and U.S. Publication No.03-0092643).

Further contemplated adjuvants include synthetic oligonucleotideadjuvants containing the CpG nucleotide motif one or more times (plusflanking sequences) available from Coley Pharmaceutical Group. Theadjuvant designated QS21, available from Aquila Biopharmaceuticals,Inc., is an immunologically active saponin fractions having adjuvantactivity derived from the bark of the South American tree QuillajaSaponaria Molina (e.g. Quil™ A), and the method of its production isdisclosed in U.S. Pat. No. 5,057,540. Derivatives of Quil™ A, forexample QS21 (an HPLC purified fraction derivative of Quil™ A also knownas QA21), and other fractions such as QA17 are also disclosed.Semi-synthetic and synthetic derivatives of Quillaja Saponaria Molinasaponins are also useful, such as those described in U.S. Pat. No.5,977,081 and U.S. Pat. No. 6,080,725. The adjuvant denominated MF59available from Chiron Corp. is described in U.S. Pat. No. 5,709,879 andU.S. Pat. No. 6,086,901.

Muramyl dipeptide adjuvants are also contemplated and includeN-acetyl-muramyl-L-threonyl-D-isoglutamine (thur-MDP),N-acetyl-nor-muramyl-L-alanyl-D-isoglutamine (CGP 11637, referred to asnor-MDP), andN-acetylmuramyl-L-alanyl-D-isoglutaminyl-L-alanine-2-(1′-2′-dipalmityol-s-n-glycero-3-hydroxyphosphoryloxy)ethylamine ((CGP) 1983A, referred to as MTP-PE). The so-called muramyldipeptide analogues are described in U.S. Pat. No. 4,767,842.

Other adjuvant mixtures include combinations of 3D-MPL and QS21 (EP 0671 948 B1), oil-in-water emulsions comprising 3D-MPL and QS21 (WO95/17210, PCT/EP98/05714), 3D-MPL formulated with other carriers (EP 0689 454 B1), QS21 formulated in cholesterol-containing liposomes (WO96/33739), or immunostimulatory oligonucleotides (WO 96/02555). AdjuvantSBAS2 (now AS02) available from SKB (now Glaxo-SmithKline) contains QS21and MPL in an oil-in-water emulsion is also useful. Alternativeadjuvants include those described in WO 99/52549 and non-particulatesuspensions of polyoxyethylene ether (UK Patent Application No.9807805.8).

The use of an adjuvant that contains one or more agonists for toll-likereceptor-4 (TLR-4) such as an MPL® adjuvant or a structurally relatedcompound such as an RC-529® adjuvant or a Lipid A mimetic, alone oralong with an agonist for TLR-9 such as a non-methylated oligodeoxynucleotide-containing the CpG motif is also optional.

Another type of adjuvant mixture comprises a stable water-in-oilemulsion further containing aminoalkyl glucosamine phosphates such asdescribed in U.S. Pat. No. 6,113,918. Of the aminoalkyl glucosaminephosphates the molecule known as RC-529{(2-[(R)-3-tetradecanoyloxytetradecanoylamino]ethyl2-deoxy-4-O-phosphono-3-O—[(R)-3-tetradecanoyloxy-tetradecanoyl]-2-[(R)-3-tetradecanoyloxytetra-decanoylamino]-p-D-glucopyranosidetriethylammonium salt.)} is preferred. One particular water-in-oilemulsion is described in WO 99/56776.

Adjuvants are utilized in an adjuvant amount, which can vary with theadjuvant, host animal and immunogen. Typical amounts can vary from about1 μg to about 1 mg per immunization. Those skilled in the art know thatappropriate concentrations or amounts can be readily determined.

Vaccine compositions comprising an adjuvant based on oil in wateremulsion is also included within the scope of the present invention. Thewater in oil emulsion may comprise a metabolisable oil and a saponin,such as for example as described in U.S. Pat. No. 7,323,182.

According to several embodiments, the vaccine compositions according tothe present invention may contain one or more adjuvants, characterizedin that it is present as a solution or emulsion which is substantiallyfree from inorganic salt ions, wherein said solution or emulsioncontains one or more water soluble or water-emulsifiable substanceswhich is capable of making the vaccine isotonic or hypotonic. The watersoluble or water-emulsifiable substances may be, for example, selectedfrom the group consisting of: maltose; fructose; galactose; saccharose;sugar alcohol; lipid; and combinations thereof.

The compositions of the present invention comprise according to severalspecific embodiments a proteosome adjuvant. The proteosome adjuvantcomprises a purified preparation of outer membrane proteins ofmeningococci and similar preparations from other bacteria. Theseproteins are highly hydrophobic, reflecting their role as transmembraneproteins and porins. Due to their hydrophobic protein-proteininteractions, when appropriately isolated, the proteins formmulti-molecular structures consisting of about 60-100 nm diameter wholeor fragmented membrane vesicles. This liposome-like physical stateallows the proteosome adjuvant to act as a protein carrier and also toact as an adjuvant.

The use of proteosome adjuvant has been described in the prior art andis reviewed by Lowell GH in “New Generation Vaccines”, Second Edition,Marcel Dekker Inc, New York, Basel, Hong Kong (1997) pages 193-206.Proteosome adjuvant vesicles are described as comparable in size tocertain viruses which are hydrophobic and safe for human use. The reviewdescribes formulation of compositions comprising non-covalent complexesbetween various antigens and proteosome adjuvant vesicles which areformed when solubilizing detergent is selectably removed usingexhaustive dialysis technology.

The present invention also encompasses within its scope the preparationand use of DNA vaccines. Vaccination methods and compositions of thistype are well known in the art and are comprehensively described in manydifferent articles, monographs and books (see, for example, chapter 11of “Molecular Biotechnology: principles and applications of recombinantDNA” eds. B. R. Glick & J. J. Pasternak, ASM Press, Washington, D.C.,2^(nd) edition, 1998). In principle, DNA vaccination is achieved bycloning the cDNAs for the desired immunogen into a suitable DNA vaccinevector, such as the pVAC vector (Invivogen), using codons optimized forexpression in human. In the case of pVAC, the desired immunogenicproteins are targeted and anchored to the cell surface by cloning thegene of interest in frame between the IL2 signal sequence and theC-terminal transmembrane anchoring domain of human placental alkalinephosphatase. The use of other immune enhancers, including adjuvants orcloning in frame other immune enhancing cytokines, together with the DNAvaccines is also within the scope of the present invention. Such DNAvaccine vectors are specifically designed to stimulate humoral immuneresponses by intramuscular injection. The antigenic peptide produced onthe surface of muscle cells is taken up by antigen presenting cells(APCs), processed and presented to the immune system T helper cellsthrough the major histocompatibility complex (MHC) class II molecules.

Oral liquid preparations may be in the form of, for example, aqueous oroily suspension, solutions, emulsions, syrups or elixirs, or may bepresented dry in tablet form or a product for reconstitution with wateror other suitable vehicle before use. Such liquid preparations maycontain conventional additives such as suspending agents, emulsifyingagents, non-aqueous vehicles (which may include edible oils), orpreservative.

The aforementioned adjuvants are substances that can be used to augmenta specific immune response. Normally, the adjuvant and the compositionare mixed prior to presentation to the immune system, or presentedseparately, but into the same site of the subject being vaccinated.Adjuvants that may be usefully employed in the preparation of vaccinesinclude: oil adjuvants (for example, Freund's complete and incompleteadjuvants, that will be used in animal experiments only and is forbiddenfrom use in humans), mineral salts, alum, silica, kaolin, and carbon,polynucleotides and certain natural substances of microbial origin. Anadditional mode of antigen delivery may include an encapsulationtechnique, which involves complex coacervation of gelatin andchondroitin sulfate (Azhari R, Leong K W. 1991. Complex coacervation ofchondroitin sulfate and gelatin and its use for encapsulation and slowrelease of a model protein. Proc. Symp. Control. Rel. 18: 617; Brown KE, Leong K, Huang C H, Dalal R, Green G D, Haimes H B, Jimenez P A,Bathon J. 1998. Gelatin/chondroitin 6-sulfate microspheres for deliveryof therapeutic proteins to the joint. Arthritis Rheum 41: 2185-2195).

Further examples of materials and methods useful in the preparation ofvaccine compositions are well known to those skilled in the art. Inaddition, further details may be gleaned from Remington's PharmaceuticalSciences, Mack Publishing Co, Easton, Pa., USA, 20^(th) edition 2000.

The S. pneumoniae cell-wall and/or cell-membrane proteins for use inworking the present invention may be obtained by directly purifying saidproteins from cultures of S. pneumoniae by any of the standardtechniques used to prepare and purify cell-surface proteins. Suitablemethods are described in many biochemistry text-books, review articlesand laboratory guides, including inter alia “Protein Structure: apractical approach” ed. T. E. Creighton, IRL Press, Oxford, UK (1989).

However, it is to be noted that such an approach suffers many practicallimitations that present obstacles for producing commercially-viablequantities of the desired proteins. The S. pneumoniae proteins of thepresent invention may therefore be more conveniently prepared by meansof recombinant biotechnological means, whereby the gene for the S.pneumoniae protein of interest is isolated and inserted into anappropriate expression vector system (such as a plasmid or phage), whichis then introduced into a host cell that will permit large-scaleproduction of said protein by means of, for example, overexpression.

As a first stage, the location of the genes of interest within the S.pneumoniae genome may be determined by reference to a complete-genomedatabase such as the TIGR database that is maintained by the Institutefor Genomic Research. The selected sequence may, where appropriate, beisolated directly by the use of appropriate restriction endonucleases,or more effectively by means of PCR amplification. Suitable techniquesare described in, for example, U.S. Pat. Nos. 4,683,195, 4,683,202,4,800,159, 4,965,188, as well as in Innis et al. eds., PCR Protocols: Aguide to method and applications. Alternatively, the gene may bechemically synthesized with codons optimized to the expression systemactually used (i.e. E. coli). For DNA vaccines, codons are optimized forexpression in human.

Following amplification and/or restriction endonuclease digestion, thedesired gene or gene fragment is ligated either initially into a cloningvector, or directly into an expression vector that is appropriate forthe chosen host cell type. In the case of the S. pneumoniae proteins,Escherichia coli is the most useful expression host. However, many othercell types may be also be usefully employed including other bacteria,yeast cells, insect cells and mammalian cell systems known in the art.

High-level expression of the desired protein (as intact proteinsequence, modified protein sequence, fragment of thereof), within thehost cell may be achieved in several different ways (depending on thechosen expression vector) including expression as a fusion protein (e.g.with factor Xa or thrombin), expression as a His-tagged protein, dualvector systems, expression systems leading to incorporation of therecombinant protein inside inclusion bodies etc. The recombinant proteinwill then need to be isolated and purified from the cell membrane,interior cellular soluble fraction, inclusion body or (in the case ofsecreted proteins) the culture medium, by one of the many methods knownin the art.

All of the above recombinant DNA and protein purification techniques arewell known to all skilled artisans in the field, the details of saidtechniques being described in many standard works including “Molecularcloning: a laboratory manual” by Sambrook, J., Fritsch, E. F. &Maniatis, T., Cold Spring Harbor, N.Y., 2^(nd) ed., 1989, which isincorporated herein by reference in its entirety.

As disclosed and explained hereinabove, each of the abovementionedembodiments of the invention may be based on the use of one or moreintact, full length, cell wall and/or cell membrane proteins or, in thealternative, or in addition thereto, fragments, derivatives andmodifications of said full length proteins. Fragments may be obtained bymeans of recombinant expression of selected regions of the cell wallprotein gene(s). Derivatives of the full length proteins or fragmentsthereof may be obtained by introducing non-native sequences within theDNA sequences encoding said proteins, followed by expression of saidderivatized sequences. Derivatives may also be produced by conjugatingnon-native groups to the amino residue side chains of the cell wallproteins or protein fragments, using standard protein modificationtechniques. Modified cell wall proteins and protein fragments for use inthe present invention may also be obtained by the use of site-directedmutagenesis techniques. Such techniques are well known in the art andare described, for example, in “Molecular cloning: a laboratory manual”by Sambrook, J., Fritsch, E. F. & Maniatis, T., Cold Spring Harbor,N.Y., 2^(nd) ed., 1989. Of particular interest is the use of one or moreof the preceding techniques to create fragments or derivativespossessing the desired epitopic sites, but lacking other domains whichare responsible for adverse effects such as suppression of cellularimmune responses. It is to be emphasized that all of the immediatelypreceding discussion of fragments, derivatives and mutants of the cellwall proteins disclosed herein are to be considered as an integral partof the present invention.

S. pneumoniae infections are common in children under the five years ofage mainly under two years of age. The infants' antibody production isknown to be produced at 6 months of age. The present invention is basedin part on a study performed with sera obtained longitudinally fromchildren at 18, 30 and 42 months of age, attending day care centers,which are exposed to the bacteria. The children's sera were screened forchange, with age, of the presence or amount of antibodies to specificcell wall/membrane proteins. Antibodies to specific proteins which wereabsent or low in sera of younger children and appear or increase withage identified proteins that now would be considered as candidate forvaccine development for protecting infants against S. pneumoniae.Without wishing to be bound to any theory it is suspected that theimmune response of younger children to the proteins in the context ofthe bacterium is also not efficient. Since the increase in the responseto these proteins is in reciprocal correlation with disease it wasassumed that immunization with these proteins will elicit a protectiveimmune response. Each of the proteins in the set disclosed for the firsttime in the present application as being associated with age-dependentimmune response to the bacteria may elicit protective immune responseagainst the bacteria at all ages to all subjects, including infants,elderly and immunocompromised subjects.

PPP enzymatic function occurs in the cytoplasm, however, it was foundalso to localize to the cell-wall and to the cytoplasmic membrane. FabD,an enzyme that is involved in lipid metabolism, could be found in thecytoplasm only but could not be found in the cell wall, furthersuggesting that under the experimental conditions used the cell-walllocalization of PPP does not result from a non-specific leakage.Moreover, live unencapsulated bacteria could be stained with an anti-PPPmonoclonal antibody, further suggesting that PPP is cell-wall localized.Membrane localization of PPP observed in immunoblots may result from itsintracellular enzymatic activity in the PTS system, which occurs near toor at the inner leaflet of the cytoplasmic membrane.

The cell-wall residence, age-dependent immunogenicity, conservationamong pneumococcal strains and adhesin activity support the vaccinepotential of PPP. Immunization with rPPP reduced nasopharyngeal and lungcolonization and reduced mortality upon challenge.

The observations that PPP resides in the cell-wall, demonstratesage-dependent antigenicity, and inhibits adhesion suggest that it couldbe a candidate vaccine antigen.

EXAMPLES

The following examples are provided for illustrative purposes and inorder to more particularly explain and describe the present invention.The present invention, however, is not limited to the particularembodiments disclosed in the examples.

Example 1

Prevention of S. pneumoniae Infection in Mice by Inoculation with S.pneumoniae Cell Wall Protein Fractions

Methods:

Bacterial Cells: The bacterial strain used in this study was an S.pneumoniae serotype 3 strain and R6. The bacteria were plated ontotryptic soy agar supplemented with 5% sheep erythrocytes and incubatedfor 17-18 hours at 37° C. under anaerobic conditions. The bacterialcells were then transferred to Todd-Hewitt broth supplemented with 0-5%yeast extract and grown to mid-late log phase. Bacteria were harvestedand the pellets were stored at −70° C.

Purification of Cell Wall Proteins: Bacterial pellets were resuspendedin phosphate buffered saline (PBS). The resulting pellets were thentreated with mutanolysin to release cell wall components. Supernatantscontaining the CW proteins were then harvested. Subsequently, thebacteria were sonicated, centrifuged and the resulting pellet containingthe bacteria membranes (m) were lysed with 0.5% TRITON™ X-100.

Fractionation of the Cell Wall Protein Mixture: Cell wallprotein-containing supernatants were allowed to adhere to fetuin (ahighly glycosylated pan-lectin binding protein) that was covalentlybound to a sepharose column. Non-adherent molecules, obtained from theflow-through fraction were predominantly non-lectin molecules, while thecolumn-adherent lectins were eluted with 50 mM ammonium acetate at pH3.5.

Experimental: S. pneumoniae cell wall (CW) proteins were separated intolectin (CW-L) and non-lectin (NL) fractions by fetuin affinitychromatography, as described hereinabove. C57BL/6 and BALB/c mice werevaccinated with S. pneumoniae total CW (CW-T), CW-L and CW-NL proteinpreparations mixed with Freund's adjuvant, by means of the followingprocedure: each mouse was primed with 25 micrograms of CW-T, CW-NL andCW-L protein preparations intramuscularly, with complete Freund'sadjuvant (CFA) and boosted with incomplete Freund's adjuvant (IFA), 4and 7 weeks following priming. Western blots of the abovementionedprotein preparations were probed with sera obtained a week after thelast immunization. Animals were then challenged intranasally (IN) orintraperitoneally (IP) with 10⁸ cfu of S. pneumoniae serotype 3, thatcaused 100% mortality in control mice immunized with CFA and boostedwith IFA only within 96 hours post-inoculation. Vaccination with CW-Lelicited partial protection against S. pneumoniae IN and IP challenge(50% and 45% respectively). Vaccination with CW-T and CW-NL proteinselicited 70% and 65% protection against IP challenge, respectively.Vaccination with CW-T and CW-NL proteins elicited 85% and 50% protectionagainst IN inoculation, respectively.

Example 2 Determination of Age-Related Immunoreactivity to S. pneumoniaeSurface Proteins

The following study was carried out in order to investigate theage-related development of immunoreactivity to S. pneumoniae cell walland cell membrane proteins.

Operating as described hereinabove in Example 1, a fraction containingcell wall proteins was obtained from a clinical isolate of S.pneumoniae. In addition, cell membrane proteins were recovered bysolubilizing the membrane pellet in 0.5% TRITON™ X-100. The cell walland cell membrane proteins were separated by means of two-dimensionalgel electrophoresis, wherein the proteins were separated usingpolyacrylamide gel isoelectric focusing in one dimension, and sodiumdodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) in theother dimension. The separated proteins were either transferred to anitrocellulose membrane or directly stained with COOMASSIE BRILLIANTBLUE™.

Sera were collected longitudinally from healthy children attendingday-care centers at 18, 30 and 42 months of age. Starting at 12 monthsof age, nasopharyngeal swabs were taken from the children on a bimonthlyschedule over the 2.5 years of the study. Pneumococcal isolates werecharacterized by inhibition with optochin and a positive slideagglutination test (Phadebact, Pharmacia Diagnostics). In addition, serawere collected from healthy adults.

The ability of serum prepared from the above-mentioned blood samples torecognize the separated S. pneumoniae proteins was investigated byWestern blot analysis according to the methods described by Rapola S. etal. (J. Infect. Dis., 2000, 182: 1146-52). Putative identification ofthe separated protein spots obtained following the 2D-electrophoresiswas achieved by the use of the Matrix Assisted LaserDesorption/Ionization mass spectrometry (MALDI-MS). The results of theabove analysis are summarized in the following table:

TABLE 1 Age-dependent immunoreactivity to S. pneumoniae surface proteinsSpot Proteins/ Age (years) no. spot Homology to 1.5 2.5 3.5 adult 1 2DNA K/phosphoenolpyruvate protein * * * * Phosphoesterase 3 1 Triggerfactor * * * * 4 2 60 KDa chaperonin (GroEl protein) Eleongation factor** * ** *** G/tetracycline resistance protein teto (TET(O)) 7 2Glutamyl-tRNA amidotransferase subunit * ** * * * A/N utilizationsybstance protein protein A 11 2 Oligopeptide-binding proteinamiA/aliA/aliB precursor Hypothetical zinc metalloproteinase in SCAA 5′region (ORF 6) 12 1 Pneumolysin (thiol-activated cytolysin * * ** 13 1L-lactate dehydrogenase * ** * 14 1 Glyceraldehyde 3-phosphatedehydrogenase * ** *** *** (GAPDH) 15 1 Fructose-bisphosphate aldolase** *** *** *** 16 1 UDP-glucose 4-epimerase ** * 17 2 Elongation factorG/tetracycline resistance * ** protein teto (TET(O)) 18 1 Pyruvatoxidase *** *** *** *** 22 1 Glutamyl-tRNA synthetase * ** 23 1NADP-specific glutamate dehydrogenase * * * 24 1 Glyceraldehydes3-phosphate dehydrogenase * ** *** **** (GAPDH) 25 1 Enolase(2-phosphoglycerate dehydratase) * ** ** ** 27 1 Phosphoglyceratekinase * ** ** ** 29 1 Glucose-6-phosphate isomerase * * ** 30 2 40Sribosomal protein S1/6-phosphogluconate dehydrogenase 31 1Aminopeptidase C 33 Carbamoyl-phosphate synthase * ** *** 57/65Aspartate carbamoyltransferase * * ** ** 58 30S ribosomal protein S2 ***

The data presented in the preceding table indicate that there is anage-dependent development of immunoreactivity to several S. pneumoniaecell wall and cell surface proteins.

Ling et al. (Clin. Exp. Immunol. 138:290-298, 2004) further describesidentification of S. pneumoniae vaccine candidates. As shown in table 2,it was found that the antigenic proteins from the enriched cell wallextract fell into three groups. The first group comprised proteins withlow immunogenicity. The second group consists of antigens for which theimmunogenicity seemed to increase with age of children attendingday-care centers, while the third group of proteins was highly antigenicwith all sera tested. The existence of serum antibodies to a certainbacterial protein does not necessarily indicate their capacity to elicitprotective immune response against the bacteria. However, the increasein the antibody response to bacterial proteins which coincides with thediminution in morbidity described in children encouraged to test theseantigens for their ability to elicit protection against S. pneumoniae.It is concluded that the immunogenic enzymes with an age dependentincrease in antigenicity of S. pneumoniae found in enriched cell walland membrane extract may represent a novel class of vaccine candidates.As shown herein for the first time many of these identifiedproteins/enzymes elicit protective level immune responses in mice andafford significant protection against respiratory challenge withvirulent S. pneumoniae.

TABLE 2 Identification of S. pneumoniae surface proteins withage-dependent immunogenicity Immunoreactivity MALDI-TOF analysis Age(months) Spot Homology Acc. number Mascot MW pI 1.5 2.5 3.5 AdultProteins with low immunogenicity 1 DNA K NP_345035 173 64.8 4.6 * * 23NADP-specific NP_345769 186 49 5.3 * * * glutamate dehydrogenaseProteins with increased immunogenicity 7 Glutamyl-tRNA NP_344959 83 524.9 * ** ** *** Amidotransferase subunit A 13 L-lactate dehydrogenaseNP_345686 134 35.9 5.2 * ** * ** 14 Glyceraldehyde 3- NP_346439 350 37.15.7 * ** *** *** phosphate dehydrogenase 15 Fructose-bisphosphateNP_345117 106 31.5 5 ** *** *** *** aldolase 16 UDP-glucose 4- NP_346051116 37.5 4.8 ** * * ** epimerase 22 Glutamyl-tRNA NP_346492 194 56 4.9 *** ** synthetase 27 Phosphoglycerate kinase NP_345017 109 41.9 4.9 * **** ** 29 Glucose-6-phosphate NP_346493 96 51.3 5.2 * * ** isomerase 306-phosphogluconate NP_344902 58 53.7 4.9 ** ** dehydrogenase 31Aminopeptidase C NP_344819 120 33.7 4.8 ** ** x Hypothetical proteinNP_358083 15 5.2 * ** 33 Carbamoyl-phosphate NP_345739 230 116.5 4.8 *** *** synthase 65 Aspartate NP_345741 44 34.7 5.1 * * ** **carbamoyltransferase Proteins with high immunogenicity 18 Pyruvateoxidase NP_345231 168 65.3 5.1 *** *** *** *** 25 Enolase (2- NP_345598215 47.1 4.7 ** ** ** ** phosphoglycerate dehydratase)

The extent of surface protein recognition by the sera was determined bythe optical density as measured by the imager used in our study(αInnotech). *Low; **intermediate; ***high

Example 3 Prevention of S. pneumoniae Infection in Mice withRecombinantly-Expressed S. pneumoniae Cell Surface Proteins

Glycolytic enzymes associated with the cell surface of Streptococcuspneumoniae are antigenic in humans and elicit protective immuneresponses in the mouse.

The glycolytic enzymes fructose-bisphosphate aldolase (FBA,NP_(—)345117, SEQ ID NO: 13), and Glyceraldehide 3 phosphatedehydrogenase (GAPDH, NP 346439, SEQ ID NO:12), which are associatedwith the cell surface of S. pneumoniae, were used to immunize miceagainst S. pneumonia as described in Ling et al., Clin. Exp. Immunol.138:290-298. 2004. It was shown that both proteins, which are antigenicin humans, elicit cross-strain protective immunity in mice.

Cloning of Immunogenic S. pneumoniae Surface Proteins: S. pneumoniaefructose-bisphosphate aldolase (hereinafter referred to as “aldolase”)and GAPDH proteins were cloned into the pHAT expression vector (BDBiosciences Clontech, Palo Alto, Calif., USA; HAT Vectors encodepolyhistidine epitope tag in which the 6 histidine are not consecutive:Lys Asp His Leu Ile His Asn Val His Lys Glu His Ala His Ala His Asn Lys(SEQ ID NO:36)), and expressed in E. coli BL21 cells (Promega Corp.,USA) using standard laboratory procedures. Following lysis of the BL21cells, recombinant proteins were purified by the use of immobilizedmetal affinity chromatography (IMAC) on Ni-NTA columns (Qiagen) andeluted with imidazole. In a separate set of experiments, S. pneumoniaealdolase cDNAs were cloned into the pVAC expression vector (Invivogen),a DNA vaccine vector specifically designed to stimulate a immuneresponse by intramuscular injection. Antigenic proteins are targeted andanchored to the cell surface by cloning the gene of interest in framebetween the IL2 signal sequence and the C-terminal transmembraneanchoring domain of human placental alkaline phosphatase. The antigenicpeptide produced on the surface of muscle cells is taken up by antigenpresenting cells (APCs) and processed to be presented to the T helpercells by the major histocompatibility complex (MHC) class II molecules.

Immunization: BALB/c and C57BL/6 mice (7 week old females) wereintraperitonealy immunized with 25 micrograms of either recombinantaldolase or recombinant GAPDH proteins together with either Freund'scomplete adjuvant (CFA) or an alum adjuvant. In a separate set ofexperiments, mice of the aforementioned strains were intramuscularlyimmunized with 50 micrograms of the pVAC-aldolase or pVAC-GAPDHconstructs that were described hereinabove.

Assessment of Immunogenicity: The immunogenicity of recombinant S.pneumoniae aldolase and GAPDH proteins was assessed by Western blotassay using serum of mice that had been immunized with either total cellwall proteins (CW-T) or with one of the recombinant proteins (asdescribed hereinabove). The results obtained (FIG. 1) indicate that thesera of the immunized animals recognized both recombinant GAPDH andaldolase proteins, and the native GAPDH and aldolase proteins present inthe CW-T mixture.

In a separate set of experiments the serum of mice that had beenimmunized with DNA vaccines of pVAC-aldolase or pVAC-GAPDH constructs,as described above, was used to detect native aldolase and GAPDH,respectively in Western blots obtained from SDS-PAGE separations of CW-Tproteins. The results obtained (FIG. 2) indicate that inoculation withthe DNA vaccines containing pVAC-based constructs is capable ofeliciting an immune response. Sera of mice vaccinated with the parentalpVAC plasmid (i.e. without insert) did not react with the CW-T proteins.

Protective Vaccination: Following immunization with the recombinantproteins as described hereinabove, the mice were challenged intranasallywith a lethal dose of 10⁸ CFU of S. pneumoniae serotype 3. Only 10% ofthe control animals (immunization with either CFA or alum only) survivedthe bacterial challenge. However, 40% of the animals immunized with therecombinant aldolase protein in CFA and 43% of the animals immunizedwith the same protein in alum survived the challenge. In contrast,immunization with the protein DNA K, having low immugenicity (table 2)did not elicit a protective immune response. Following immunization withthe pVAC-aldolase construct, 33% of the animals survived. With regard torecombinant GAPDH, 36% of the animals immunized with this recombinantprotein survived. Immunization with the pVAC-GAPDH construct, led to asurvival rate of 40%, as shown in FIG. 3.

Example 4 S. pneumoniae Immunogenic Proteins

Operating essentially as in Example 2, the ability of serum preparedfrom blood samples of children aged 1.5, 2.5 and 3.5 years and adults torecognize the separated S. pneumoniae proteins was investigated byWestern blot analysis according to the methods described by Rapola S. etal. (J. Infect. Dis., 2000, 182: 1146-52).

Identification of the separated protein spots obtained following the2D-electrophoresis was achieved by the use of the Matrix Assisted LaserDesorption/Ionization mass spectrometry (MALDI-MS) technique, andcomparison of the partial amino acid sequences obtained thereby with thesequences contained in the TIGR4 and/or R6 databases (maintained by TheInstitute for Genomic Research).

The cell surface proteins found to be immunogenic (classified accordingto their cellular location—cell membrane or cell wall) are summarized inthe following table:

TABLE 3 list of immunogenic proteins Accession SEQ Spot # Protein nameNo. ID NO 1 phosphoenolpyruvate protein phosphotransferase NP_345645 4 2phosphoglucomutase/phosphomannomutase family NP_346006 5 protein 3trigger factor NP_344923 6 4 elongation factor G/tetracycline resistanceprotein NP_344811 7 (tetO) 6 NADH oxidase NP_345923 8 7Aspartyl/glutamyl-tRNA amidotransferase subunit C NP_344960 9 8 celldivision protein FtsZ NP_346105 10 13 L-lactate dehydrogenase NP_34568611 14 glyceraldehyde 3-phosphate dehydrogenase (GAPDH) NP_346439 12 15fructose-bisphosphate aldolase NP_345117 13 16 UDP-glucose 4-epimeraseNP_346261 14 elongation factor Tu family protein NP_358192 15 21Bifunctional GMP synthase/glutamine amidotransferase NP_345899 16protein 22 glutamyl-tRNA synthetase NP_346492 17 23 glutamatedehydrogenase NP_345769 18 26 Elongation factor TS NP_346622 19 27phosphoglycerate kinase (TIGR4) AAK74657 20 30 30S ribosomal protein S1NP_345350 21 6-phosphogluconate dehydrogenase NP_357929 22 31aminopeptidase C NP_344819 23 33 carbamoyl-phosphate synthase (largesubunit) NP_345739 24 57 PTS system, mannose-specific IIAB componentsNP_344822 25 58 30S ribosomal protein S2 NP_346623 26 62 dihydroorotatedehydrogenase 1B NP_358460 27 65 aspartate carbamoyltransferasecatalytic subunit NP_345741 28 14 elongation factor Tu NP_345941 29 19Pneumococcal surface immunogenic protein A (PsipA) NP_344634 30 22phosphoglycerate kinase (R6) NP_358035 31 40 ABC transportersubstrate-binding protein NP_344690 32 10 endopeptidase O NP_346087 3314 Pneumococcal surface immunogenic protein B (PsipB) NP_358083 34Pneumococcal surface immunogenic protein C (PsipC) NP_345081 35

Example 5 Preparation of an S. pneumoniae Fructose Bisphosphate AldolaseFragment

A peptide referred to as ALDO 1, corresponding to the first 294nucleotides of the coding sequence of the fructose bisphosphate aldolasegene (SP0605 Streptococcus pneumoniae TIGR4) (SEQ ID NO:1), wasamplified from S. pneumoniae strain R6 genomic DNA by means of PCR withthe following primers:

 (SEQ ID NO: 2)  3 Forward (5′-GGT ACC ATG GCA ATC GTT TCA GCA-3′),(SEQ ID NO: 3) Reverse (5′-GAG CTC ACC AAC TTC GAT ACA CTC AAG-3′). 

The amplified product obtained thereby is shown in FIG. 4.

The Forward and Reverse primers, constructed according to the TIGR4sequence contain Kpn1 and SacI recognition sequences, respectively. Theprimers flank the entire open reading frames.

The primers were used to amplify the gene from S. pneumoniae serotype 3strain WU2. The amplified and Kpn1-SacI (Takara Bio Inc, Shiga, Japan)digested DNA-fragments were cloned into the pHAT expression vector (BDBiosciences Clontech, Palo Alto, Calif., USA; as described in Example3), as illustrated in FIG. 5 and transformed in DH5a UltraMAXultracompetent E. coli cells.

Ampicillin-resistant transformants were cultured and plasmid DNA wasanalyzed by PCR. The pHAT-ALDO 1 vector was purified from DH5.alpha.UltraMAX cells using the Qiagen High Speed Plasmid Maxi Kit (QiagenGMBH, Hilden, Germany) and transformed in E. coli host expression strainBL21(DE3)pLysS. PCR amplification of the ALDO 1 fragment fromtransformed positive colonies yielded the 297 by fragment indicated inthe gel shown in FIG. 6.

Example 6 Cloning, Expressing and Purification of RecombinantPhosphoenolpyruvate Protein Phosphotransferase (PPP) Proteins

Two genetically unrelated encapsulated S. pneumoniae strains, serotype 2strain D39 (Avery 1995, Mol Med 1: 344-365) and serotype 3 strain WU2(Briles 1981, J Exp Med 153: 694-705) were used together with theirunencapsulated derivatives, strain R6 (ATCC, Rockville Md.) and strain3.8DW (Watson at al., 1990, Infect Immun 58: 3135-3138). Pneumococciwere grown in THY or on blood agar plates as previously described(Mizrachi Nebenzahl, et al., 2004, FEMS Microbiol Lett 233: 147-152).Two Escherichia coli strains were used, DH5α UltraMAX (DH5α; InvitrogenCorp, Carlsbad, Calif., USA) and BL21(DE3)pLysS (BL21; Promega Corp,Madison, Wis., USA) and were grown in lysogeny broth (LB).

The nucleotide sequence of the NP_(—)345645 PPP protein was amplifiedfrom pneumococcal serotype 3 strain WU2 genomic DNA according to thepublished sequence of serotype 4 strain TIGR4 by PCR with the followingprimers:

Forward: 5′-GGATCCATGACAGAAATGCTTAAAG-3′ (SEQ ID NO:36) and Reverse5′-GAGCTCTTAATCAAAATTAACGTATTC-3′ (SEQ ID NO:37) (supplemented withrestriction enzyme sequences of BamHI (Takara Biomedicals, Otsoshiga,Japan) on the 3′ end and Sac1 on the 5′ end (Takara Biomedicals,Otsoshiga, Japan). The amplified product was cloned into the pHATexpression vector (BD Biosciences Clontech, Palo Alto, Calif., USA), andprotein expression and purification were performed as previouslydescribed (Mizrachi Nebenzahl, et al., 2007 ibid). Verification ofsequence identity was performed by plasmid insert sequencing. Thetagged-purified protein was resolved by SDS-polyacrylamide gelelectrophoresis (PAGE). Pneumococcal cell-wall proteins were separatedby SDS-PAGE under reducing conditions and transferred to nitrocellulosemembranes (Bio-Rad, Carlsbad, Calif., USA) as previously described(Ausubel F, 1989). Separation showed that the 75 kDa HAT-PPP fusionprotein was ˜95% pure. The rPPP. The identity of PPP was furtherconfirmed by immunoblot analysis using either rabbit anti-PPP antiserumor human sera. Immunoblotting with anti-HAT antibodies confirmed theidentity of the protein sequence was verified by MALDI-TOF analysis aspreviously described using a Bruker Reflex-IV mass spectrometer(Bruker-Daltonik, Bremen, Germany) (Portnoi, et al., 2006, Vaccine 24:1868-1873). MALDI-TOF analysis of this protein band identified rPPP in99% accordance with the expected PPP protein (PI=4.6, Mascot score=92, Zscore=2.43, extent of sequence coverage=39).

Immunization of Rabbits with rPPP

Three-month-old white albino rabbits (Harlan Laboratories, Israel) wereinitially immunized intramuscularly (IM) with 200 μg HAT-rPPP emulsifiedwith complete Freund's adjuvant (CFA) (1:1) in the first immunization orwith incomplete Freund's adjuvant (IFA) in booster immunizations. Twoweeks after their final immunization rabbits were exsanguinated and seraprepared.

Surface Expression and Conservation of PPP in Different PneumococcalStrains

To analyze surface expression and conservation, immunoblot analysis ofcell-wall and membrane protein fractions from several pneumococcalstrains using anti-rPPP antisera was performed. PPP was found to resideboth in the cell-wall and in the membranes of different strains (FIG.17A). The differences found in the molecular weight of PPP may resultfrom post-translation modifications. In contrast to PPP, no cell-wallresidence could be found for the rFabD protein (FIG. 17B).

Alignment of the protein sequence from the R6 strain with the publishedpneumococcal strains sequences, performed using both the Mascot softwarepackage (Matrix Science Ltd., UK) and Profound program (RockefellerUniv.), demonstrated homology with >99% identity and 100% positivitywith no gaps.

Flow cytometry analysis performed as previously described (MizrachiNebenzahl, et al., 2007 ibid) with the R6 bacteria strain probed withanti-PPP mAbs demonstrated PPP surface expression. Strain R6 bacteriawere incubated with anti-rPPP mAb or pre-immune mouse serum, washed, andstained with Alexa Fluor 647-conjugated goat-anti-mouse-IgG (JacksonImmunoResearch, West Grove, Pa.). Flow cytometry was performed using aFACSCalibur flow cytometer (Becton Dickinson, Mountain View, Calif.),and data were acquired and analyzed using BD CellQuest™ 3.3 software.

Age-Dependent Immunogenicity of PPP

In previous studies, a group of cell-wall proteins demonstratedage-dependent antigenicity in children. To test whether PPP belongs tothis group, rPPP was immunoblotted with pediatric sera. Sera werecollected longitudinally at 18, 30 and 42 months of age from healthychildren attending day-care centers. Nasopharyngeal swabs were takenfrom the children bimonthly starting at 12 months of age for the entire3.5-year duration of the study, and episodes of carriage of differentserotype strains were documented (Lifshitz, et al., 2002, Clin ExpImmunol 127: 344-353). Increased PPP antigenicity was observed at 24months relative to 7 and 12 months with variable recognition at 38months of age (FIG. 18).

Active Immunization with PPP Reduces Nasopharyngeal and LungColonization Upon Intranasal Challenge

Seven-week-old BALB/cOlaHsd (BALB/c) female mice (Harlan Laboratories,Israel) or seven-week-old CBA/CaHN-Btk^(xid)J(CBA/Nxid; JacksonLaboratories, Bar Harbor, Me., USA) mice were housed in sterileconditions under 12-h light/dark cycles and fed Purina Chow and tapwater ad libitum.

BALB/c or CBA/Nxid mice were immunized subcutaneously (SC) with 5 or 25μg rPPP or a 25-μg NL fraction as positive control (Portnoi, et al.,2006 ibid), emulsified with CFA and boosted (days 14 and 28) with IFA.One week after third immunization the mice were anesthetized with Terrelisoflurane (MINRAD, NY, USA) and inoculated intranasally (IN) on day 42with a sublethal dose (5×10⁷) of S. pneumoniae Serotype 3 strain WU2.Mice were sacrificed by cervical dislocation 3 and 48 h later, and thenasopharynx (NP) and right lobe lung were excised, homogenized andsamples were plated onto blood agar plates for bacterial enumeration.After a similar immunization regimen, BALB/c mice were challenged INwith a lethal dose (10⁸ CFU) of strain WU2, and mortality was monitoreddaily.

Mice immunized with rPPP demonstrated a significant reduction incolonization at 3 h (FIG. 7A) and 48 h (FIG. 7B) after inoculation withstrain WU2 and at 48 h (FIGS. 7C and D) after inoculation with strainD39. Immunization with rPPP reduced mortality in BALB/c following an INlethal challenge with WU2 strain (p<0.05, FIG. 7E).

Adhesion is Mediated by PPP

To analyze whether PPP is involved in pneumococcal interaction with thehost, the ability of rPPP to inhibit pneumococcal adhesion to cells wastested. A549 cells (type II epithelial lung carcinoma cells; ATCC,Rockville, Md., USA) or Detroit562 cells (pharyngeal carcinoma derivedcells; ATCC, Rockville, Md., USA) were cultured on fibronectin-coated96-well plates (2.5×10⁴ cells/well) in DMEM (without antibiotics).Experiments were conducted in triplicate with rPPP (0-600 nM) aspreviously described (Blau, et al., 2007, J Infect Dis 195: 1828-1837).Inhibition of adhesion to A549 cells by anti-rPPP antibodies was alsoperformed.

In a dose-dependent manner, rPPP significantly inhibited the adhesion ofstrain WU2 and its unencapsulated derivative strain 3.8DW and of D39 andits unencapsulated derivative strain R6. Rabbit anti-rPPP antiserasignificantly inhibited the adhesion of strains WU2 and 3.8DW. Mouseanti-rPPP antisera significantly inhibited the adhesion of strains D39and R6 in a dose-dependent manner.

Example 7 Active Immunization with Glutamyl tRNA Synthetase

Active immunization with Glutamyl tRNA synthetase (GtS, NP_(—)346492,SEQ ID NO: 17) using alum as adjuvant is described in Mizrachi et al., JInfect Dis. 196,945-53, 2007. The cloning of the gene was byamplification of the gene using primers constructed according to theTIGR4 sequence and the gene was amplified from S. pneumoniae serotype 3strain WU2. The amplified gene was inserted into the pHAT vector asdescribed in Example 3.

Thirty-nine percent of rGtS-immunized mice survived a lethal bacterialchallenge, whereas no control mice survived. The results suggested thatGtS, an age-dependent S. pneumoniae antigen, is capable of inducing apartially protective immune response against S. pneumoniae in mice.Active immunization with rGtS using CFA as adjuvant: BALB/c mice wereimmunized three times IM with 10 μg of rGtS in CFA/IFA/IFA in 3 weeksintervals. Mice were subsequently challenged with S. pneumoniae serotype3 strain WU2. Survival was monitored up to 8 days after challenge. Asdepicted in FIG. 8, sixty percent of immunized mice survived theintranasal lethal challenge as opposed to 20% of adjuvant immunized(control) mice.

Example 8 Active Immunization with NADH Oxidase (NOX)

The cloning of the gene was by amplification of the gene using primersconstructed according to the R6 sequence and the gene was amplified fromS. pneumoniae R6. The amplified gene was inserted into the pHAT vectoras described in Example 3.

BALB/c mice were IP immunized with 25 μg of rNOX protein (NP_(—)345923,SEQ ID NO: 8), 10 μg of a mixture of non-lectin (NL) proteins as apositive control and adjuvant only as a negative control. Theimmunizations were performed in the presence of CFA in the firstimmunization and IFA in the following 2 booster immunizations given intwo weeks intervals. Mice were subsequently challenged with a lethaldose of S. pneumoniae serotype 3 strain (WU2). Survival was monitoreddaily for 7 days. While only 50% of control mice survived the bacterialchallenge 100% of NL immunized and 92% of rNOX immunized mice survivedthe challenge as shown in FIG. 9.

Example 9

Passive immunization with Pneumococcal surface immunogenic protein B(PsipB; NP_(—)358083, SEQ ID NO:34). The cloning of the gene was byamplification of the gene using primers constructed according to theTIGR4 sequence and the gene was amplified from S. pneumoniae serotype 3strain WU2. The amplified gene was inserted into the pHAT vector asdescribed in Example 3.

BALB/c mice were IP passively immunized two times with 100 μl ofanti-PsipB antiserum 24 and 3 hours prior to bacterial challenge. Micewere IP challenged with S. pneumoniae strain 3 (WU2). Survival wasmonitored up to 7 days. Administration of either anti PsipB antiserum orthe anti NL antisera protected the mice (80 and 70% respectively, FIG.10) from a lethal challenge, while the control (preimmune) serum did notprotect the mice from such challenge.

Example 10 Active Immunization with Trigger Factor (TF, NP 344923, SEQID NO:6)

The cloning of the gene was by amplification of the gene using primersconstructed according to the TIGR4 sequence and the gene was amplifiedfrom S. pneumoniae strain R6. The amplified gene was inserted into thepET32a+ vector lacking the thioredoxin sequence. The vector contain a5.7kDs tag protein which contains 6 consecutive histidines.

BALB/c mice were IP immunized (three times; CFA/IFA/IFA) with 25 μg ofTF. Mice were subsequently challenged IN with S. pneumoniae serotype 3strain WU2. Survival was monitored for 21 days. 25 μg TF elicited aprotective immune response against a lethal challenge (80%) while miceimmunized with adjuvant only were not protected (19% and 23 survival,respectively, FIG. 11)

Example 11 FtsZ Cell Division Protein (NP_(—)346105, SEQ ID NO:10)

The cloning of the gene was by amplification of the gene using primersconstructed according to the TIGR4 sequence and the gene was amplifiedfrom S. pneumoniae strain R6. The amplified gene was inserted into thepET32a+ vector lacking the thioredoxin sequence. The vector contain a5.7kDs tag protein which contains 6 consecutive histidines.

BALB/c mice were IP challenged with S. pneumoniae serotype 3 strain WU2after 1 hour neutralization with rabbit anti-FtsZ antiserum, preimmuneserum or anti NL serum. Survival was followed up to 7 days. Both theanti FtsZ and the anti NL antisera protected the mice from a lethalchallenge (50% and 86%, respectively), while the preimmune serumprotected 30% of the challenged mice (FIG. 12).

Example 12 PTS System, Mannose-Specific IIAB Components NP_(—)344822,SEQ ID NO:25)

The cloning of the gene was by amplification of the gene using primersconstructed according to the TIGR4 sequence and the gene was amplifiedfrom S. pneumoniae strain R6. The amplified gene was inserted into thepET32a+ vector lacking the thioredoxin sequence. The vector contain a5.7kDs tag protein which contains 6 consecutive histidines.

BALB/c mice were IP challenged with S. pneumoniae strain 3(WU2) after 1hour neutralization with rabbit anti-PTS antiserum. Survival wasfollowed up to 7 days. Both the anti PTS and the anti NL antiseraprotected the mice from a lethal challenge (40 and 100%, respectively),while only 10% of mice survived following challenge with bacteriapretreated with preimmune serum (FIG. 13).

Example 13 Vaccination with 6-Phosphogluconate Dehydrogenase (6PGD,NP357929, SEQ ID NO:22)

Use of 6PGD for inducing protective immune response in mice wasdescribed in Daniely et al., 144:254-63. 2006. Immunization of mice withr6PGD protected 60% of mice for 5 days and 40% of the mice for 21 daysfollowing intranasal lethal challenge, while none of the control micesurvived the same challenge after four days.

Example 14 Active Immunization with Elongation Factor G (EFG, NP344811,SEQ ID NO:7)

The cloning of the gene was by amplification of the gene using primersconstructed according to the R6 sequence and the gene was amplified fromS. pneumoniae strain S. pneumoniae serotype 3 strain WU2. The amplifiedgene was inserted into the pHAT vector lacking the thioredoxin sequence.The vector contains a 5.7kDs tag protein which contains 6 consecutivehistidines.

BALB/c mice were immunized IP with 25 μg of EFG in the presence of Alum.Mice were subsequently challenged IN with S. pneumoniae serotype 3strain WU2. Survival was monitored for 21 days. As shown in FIG. 14, EFGelicited a protective immune response against a lethal challenge in 30%of the mice, while all control mice, immunized with adjuvant only,succumbed 5 days following the bacterial challenge.

Example 15 Clinical Studies

The first Phase 1 study is performed in 20-25 adults, testing thecandidate vaccine for safety and immunogenicity. The second Phase 1study evaluates 2 or 3 dosage levels of the vaccine in groups of 20-25infants each for safety and immunogenicity.

The first Phase 2 study is performed in 100-150 infants at a developedworld site using the dosage level chosen in Phase 1, and evaluatessafety and immunogenicity as well as obtain more information about apotential surrogate assay. The second Phase 2 study at a developed worldsite is performed in 300-500 in infants in multiple sites, and evaluatesinteractions with other concomitant vaccines for extended safety andimmunogenicity. The third Phase 2 study is performed in parallel 200infants at the developing world location at which the Phase 3 efficacystudy performed, to confirm immunogenicity and safety before Phase 3.

The Phase 3 efficacy study would be performed in a developing world sitein 50,000 infants as a placebo-controlled double-blind study with aclinical endpoint.

The Phase 3 immunogenicity study would be performed in parallel in adeveloped world site using 3 different lots of final manufacturing-scalevaccine in 4 groups of 200 infants each. The Phase 3 safety study wouldbe performed in parallel in 10,000 infants in developed world sites.

Example 16 Verification of Immunogenicity and Age-Dependency of Nox andGtS

To verify that GtS induces an age-dependent immune response, sera from 3healthy children attending day care centers (with documented episodes ofcarriage of different S. pneumoniae serotypes) were obtainedlongitudinally between 18-42 months of age. A representative seriesrevealing quantitative and qualitative enhancement of antibody responsesto rGtS protein over time is shown in FIG. 15. The rGtS protein wasundetected by the infants' sera at 18 and slightly detected at 30 monthsof age. Maximal detection of rGtS with the children's sera was observedat 42 months of age. Sera obtained from a healthy adult detected rGtS tothe highest extent.

Immunoblot analysis of rNOX with sera obtained longitudinally fromchildren attending day-care centers demonstrated age-dependentenhancement in protein recognition in all 3 children (FIG. 16).

While specific embodiments of the invention have been described for thepurpose of illustration, it will be understood that the invention may becarried out in practice by skilled persons with many modifications,variations and adaptations, without departing from its spirit orexceeding the bounds of the present invention.

1. A vaccine composition comprising as the active ingredient a purifiedpreparation of the cell wall protein ABC transporter substrate-bindingprotein having the Accession No. NP_(—)344690 and the amino acidsequence set forth in SEQ ID NO: 32, optionally together with one ormore pharmaceutically acceptable adjuvants.
 2. The vaccine compositionaccording to claim formulated for administration to an infant under fouryears of age.
 3. The vaccine composition according to claim 1,formulated for administration to an infant under two years of age. 4.The vaccine composition according to claim 1, formulated foradministration to an elderly subject.
 5. A vaccine compositioncomprising at least one polynucleotide sequence encoding a proteinaccording to claim 1, optionally together with one or morepharmaceutically acceptable adjuvants.
 6. The vaccine composition ofclaim 5 further comprising at least one polynucleotide sequence encodingan adjuvant peptide or protein.
 7. A method of inducing a protectiveimmune response in a mammalian subject against Streptococcus pneumoniaecomprising administering to said subject an amount of a vaccinecomposition according to claim 1, wherein the amount is effective toinduce said protective immune response in said subject againstStreptococcus pneumoniae.
 8. The method according to claim 7, whereinthe subject is an infant under four years of age.
 9. The methodaccording to claim 7, wherein the subject is an infant under two yearsof age.
 10. The method according to claim 7, wherein the subject is anelderly subject.
 11. The method according to claim 7, wherein thesubject is an immunocompromised subject.
 12. The method according toclaim 7, wherein composition includes one or more pharmaceuticallyacceptable adjuvants.
 13. The method of claim 7, wherein the subject isprotected against S. pneumonia infection by administration of thevaccine composition prior to occurrence of said infection.
 14. Thevaccine composition according to claim 1, wherein the subject is animmunocompromised subject.
 15. A vaccine composition consistingessentially of, as the active ingredient, a purified preparation of thecell wall protein ABC transporter substrate-binding protein having theAccession No. NP_(—)344690 and the amino acid sequence set forth in SEQID NO: 32, optionally together with one or more pharmaceuticallyacceptable adjuvants.
 16. The vaccine composition according to claim 14,wherein the one or more pharmaceutically acceptable adjuvants arepresent and the composition is formulated for administration to aninfant under four years of age.
 17. The vaccine composition according toclaim 14, wherein the one or more pharmaceutically acceptable adjuvantsare present and the composition is formulated for administration to aninfant under two years of age.
 18. The vaccine composition according toclaim 14, wherein the one or more pharmaceutically acceptable adjuvantsare present and the composition is formulated for administration to anelderly subject.
 19. The vaccine composition according to claim 14,wherein the one or more pharmaceutically acceptable adjuvants arepresent and the composition is formulated for administration to animmunocompromised subject.